A sequential method for removing Cr(VI) was studied using polymer-nano zero valent iron (nZVI)-based composite film and live biomass of sulfate reducing bacteria (SRB) under anaerobic conditions. The polymer-nZVI composite film was made using layer-by-layer coating of chitosan, polyethylene glycol (PEG) blend, poly (sodium 4-styrene sulphonate) solution (PSS), and nZVI on a glass slide. The conditions for the removal of Cr(VI) through polymer-nZVI nanocomposite film were optimized using Response Surface Methodology (RSM). SRB was grown in Baar's medium was adapted to 10 mg L−1 of Cr(VI) and the live biomass obtained thereof was used for removal of Cr(VI). Cr(VI) removal capacity of 394 mg g−1 was observed at optimal conditions by nano-bio sequential removal process using polymer-nZVI-based composite film and SRB. The XRD, SEM-EDX, and FT-IR spectral data demonstrated the reduction of Cr(VI) and binding of chromium (Cr) species on the polymer-nZVI-based composite film and SRB biomass. The applicability of the sequential Cr(VI) removal process using polymer-nZVI-based composite film and SRB was successfully verified in Cr(VI)-spiked environmental water samples.