Anaerobic digestion of post-tanning wastewater was performed in batch anaerobic digester to evaluate the effect of COD/sulphate ratio [0.62, 0.69, and 1.20 (w/w) %] and F/M ratio [0.2, 0.3, 0.5, 0.7, 0.9, 1.1, 1.3, and 1.5 (w/w) %)] on the removal efficiency of COD. The F/M ratio of 0.3 was found to be the optimum ratio for the removal of COD by 53, 57, and 65%, respectively at COD/sulphate ratio of 0.62, 0.69, and 1.20. The maximum sulphate removal was observed at F/M ratio of 0.2 and the removal efficiency was 48, 50, and 58% at COD/sulphate ratio of 0.62, 0.69, and 1.20, respectively. The removal efficiency of COD and sulphate was increased with increase in COD/sulphate ratio from 0.62 to 1.20 and decreased with increase in F/M ratio from 0.2 to 1.5 in anaerobic digestion of post-tanning wastewater. The maximum concentration of sulphide formation was 784 mg/L at COD/sulphate ratio of 0.62 in anaerobic digestion process and the process was inhibited at this sulphide concentration. The microbial activity in the sludge was evaluated through live and dead cell assay using fluorescent microscopy. The maximum amount of dead microbes was observed in the anaerobic digester, which was operated at COD/sulphate ratio of 0.62 than other studied ratio.