Born in 1965 in Jersey City, New Jersey, Mazurek moved with his family to the Chicago area as a preteen, and began playing cornet in his school band at the age of ten. After switching briefly to trumpet, he eventually settled on the smaller horn. Upon graduating from high school, the young musician attended the Bloom School of Jazz in Chicago. He got his start as a performer in the city, playing with pianist Kenny Prince and drummer Yoron Israel. Early influences came from the hard bop nucleus of Blue Note Records (particularly the trumpet playing of Lee Morgan, Kenny Dorham, and Freddie Hubbard). It was in this mold that Mazurek shaped his first group, a quartet with drummer George Fludas, bassist John Webber, and pianist Randolph Tressler. In 1993, Mazurek booked an engagement at Edinburgh's Tron Tavern & Ceilidh House for the entire length of the country's Fringe Festival. Though the music was fairly traditional, the performances were a success, and the following year the group was offered a deal with Scotland's Hep label. The quartet's promise was certified with Man Facing East in 1994 and Badlands in 1995.

Mazurek was hearing a new sound, however. His creative impulses were being sparked by the music of Ornette Coleman and his cornet-wielding sparring partner from the late '50s, Don Cherry, as well as the Art Ensemble of Chicago. The list would quickly grow to include the free playing of Henry Threadgill, Bill Dixon, and Leo Smith, and by the time of 1996's Green & Blue, Mazurek was beginning to see the limitations of his quartet. That year he established the Chicago Underground, a Sunday afternoon workshop at a local bar, the Green Mill. The first person he invited to his new musical high-wire act was guitarist Jeff Parker. The jam sessions quickly attracted a group of regulars including bassist Noel Kupersmith and drummer Chad Taylor, and Mazurek found that he had the core for an ensemble. It was this new crew (completed by trombonist Sara Smith), billed as the Chicago Underground Orchestra, that recorded Playground for Bob Koester's Delmark in 1998. The partnership automatically linked Mazurek's group with a rich tradition of Chicago jazz, as Delmark had championed a number of artists from the city's Association for the Advancement of Creative Musicians.

Mazurek's group would spawn a number of spin-offs including a Duo (with Taylor), Trio (with Kupersmith), and a Quartet (with Parker), all of which wore the Chicago Underground banner. Parker had moved into the loft of Chicago out-rockers Tortoise a few years earlier and quickly became a full-time member. Two musical worlds -- fringe jazz and fringe rock -- met and Mazurek found himself exposed to an entirely new realm of sonic possibilities. In the years that followed, he made numerous extracurricular appearances, lending his cornet to Tortoise's TNT, Gastr del Sol's Camoufleur, Stereolab's Cobra and Phases Group, and Sam Prekop's solo debut, among others. It was out of this musical cross-fertilization that, in 1997, Isotope 217 was born. A disorienting mix of jazz flourishes, muscular funk, hip-hop reductions, and minimalist electronica, the group was a collaboration between Chicago Underground members (Mazurek, Parker, and Smith), veteran Chicago bassist Matt Lux, and Tortoise's Dan Bitney and John Herndon. The sextet released its debut, The Unstable Molecule, in 1998, followed by Utonian Automatic (1999) and Who Stole the I Walkman? (2000).

Meanwhile, the music emanating from the Chicago Underground was approaching the realm of pure sound on albums like Synesthesia and Possible Cube. Mazurek had discovered the music of Morton Feldman and Vladimir Ussachevsky as well as visual artists like Mark Rothko, all of which seemed to find a way into the music. As the century drew to a close, he was hard at work developing laptop-constructed musique concrète for his Orton Socket project. Mazurek subsequently moved from the United States to São Paulo, Brazil, and has continued to maintain an internationalist perspective, touring and recording with such ensembles as the São Paulo Underground, the Exploding Star Orchestra, and the improvisational trio Tigersmilk, and presenting new works at the Dieppe Biennale in Normandy, France, in 2007. In addition, the Exploding Star Orchestra appeared at the Chicago Jazz Festival with vanguard jazz legend Bill Dixon. Together they premiered one new extended composition by each man. It was the latter's first large-scale experiment with a video score. In 2008, the collaboration continued in the recording studio, resulting in Bill Dixon with Exploding Star Orchestra on Thrill Jockey. It was Dixon's final recording.

Mazurek was extremely active in 2010. He recorded Boca Negra with the Chicago Underground Duo, Stars Have Eyes with the Exploding Star Orchestra (both on Thrill Jockey), and a solo effort entitled Calma Genta, on Submarine Records' Catune imprint. In 2011, he issued Double Demon, the initial offering by his new Starlicker group, on Delmark, with vibraphonist Jason Adasiewicz and drummer John Herndon. In addition, his stellar São Paulo Underground group released its third offering, Tres Cabeças Loucuras on Cuneiform. He toured behind both. Given his other projects, the Chicago Underground Duo remained near the top of Mazurek's priority list. The pair released Age of Energy in the early spring of 2012. In January of the following year, Cuneiform released Skull Sessions by the Rob Mazurek Octet, featuring members of Starlicker, the São Paulo Underground, and other contributing musicians (including noted Chicago flutist Nicole Mitchell). In April, The Space Between, a collaborative project between his Exploding Star Electro Acoustic Ensemble and videographer Marriane F. Kim, was issued in a double-disc package containing both audio and video discs by Delmark.