The current study reports environmental application of zero-valent iron nanoparticles (NZVI)-laden anaerobic microbial granules for the removal of a major environmental pollutant, Cr (VI), from aqueous solutions. The anaerobic NZVI granules were characterized using X-ray diffraction, SEM, EDX, and FTIR analyses. For Cr (VI) removal experiments, Box–Behnken Design (BBD) with three-level factors was applied. The three operating variables, with the highest influence on Cr(VI) removal were initial concentration of Cr(VI), reaction time, and anaerobic NZVI granule dry weight. A very high removal capacity of 296.7 ± 1.82 mg/g was observed with the optimized conditions (Cr (VI) Initial concentration: 10 mg/L; anaerobic NZVI granule weight dry weight: 50 mg; reaction time: 90 min). The reduction kinetics followed pseudo-second-order model. The residual toxicity of the effluent solution after treating with the anaerobic NZVI granules was examined using microalgae, artemia, and Allium bioassays, proving a significant detoxification obtained using the process. The feasibility of using the anaerobic NZVI granules for removal of Cr (VI) from real water systems was further confirmed using groundwater and lake water samples spiked with Cr (VI). The Cr (VI) removal was noted at 88 ± 1.56 % with 231.7 ± 1.21 mg/g capacity, whereas in the lake water, they were estimated to be 79 ± 1.94 % and 217 ± 0.91 mg/g respectively, with the optimized conditions (Cr (VI) concentration added in real water system: 10 mg/L; anaerobic NZVI granule dry weight: 50 mg; reaction time: 90 min).