Heavy metals may affect anaerobic fermentation processes, which could result in variations in anaerobic fermentation parameters. In the present study, the impact of changing hexavalent chromium (Cr6+) concentrations on different anaerobic fermentation parameters was investigated. The results showed that adding Cr6+ affected fermentation efficiency since it elicited changes in enzyme activity as well as metabolic pathways. Adding Cr6+ (30 mg/L) resulted in significant positive correlation between coenzyme F420 activities, CH4 contents, and cumulative biogas yields. The results suggested Cr6+ enhanced biogas production by promoting the activity of coenzyme F420. Increasing the concentration of Cr6+ to 100 mg/L resulted in a significant correlation (P < 0.01) between the CH4 contents and cumulative biogas yields and oxidation–reduction potential and pH. At high concentrations, Cr6+ (500 mg/L) elicited parametric relationships dissimilar to those obtained using low Cr6+ concentrations, particularly on the hydrolysis stage. This study comprehensively demonstrated the influence of Cr6+ stress during the anaerobic fermentation process from a new insight and could provide critical references for further experimental design and data monitoring investigations.