The present study was planned in vitro with pearl millet stover as substrate to delineate the effect of different concentrations and sources of sulfur besides rumen fungi habitat on ruminal biochemical parameters, in buffalo and cattle. The results revealed that rumen metabolites viz. total ruminal nitrogen, ammonia nitrogen, protein-N, total volatile fatty acids and cellulase enzyme activity were significantly (P＜0.05) influenced by sulfur supplementation irrespective of habitat of rumen fungi in buffalo and cattle. Total nitrogen, protein-N and TVFA concentration in the ruminal fluid was significantly (P＜0.05) higher at 0.3% of sulfur level while ruminal fluid ammonia nitrogen concentration was lowest at same level that supports the maximum assimilation of ammonia nitrogen in to fungal protein synthesis. Organic (DL-Methionine) and inorganic (Sodium Sulfate) sources of sulfur were equally effective in promoting total nitrogen, protein-N and TVFA concentration in the ruminal fluid and at the same time surpassing effect on ammonia nitrogen production was also noticed in both ruminant species. Buffalo surpassed (P＜0.05) cattle for total ruminal nitrogen concentration though reversed situation was noticed for ammonia nitrogen though no species difference (P＜0.05) was noticed for ruminal protein-N concentration in the culture filtrate. Incubation period showed significant (P＜0.05) affect on rumen metabolites production. Fiber degrading enzyme (cellulase C1 or exo-β, 1-4 glucanase) activity increased significantly (P＜0.05) in both supplemental form of sulfur irrespective of fungi habitat and recorded highest at 0.3% sulfur level. Though no significant (P＜0.05) difference was observed for this trait due to supplemental form of sulfur and fungal habitat. The present study inferred that 0.3% sulfur supplementation helps to attain maximum fungal protein synthesis with highest catalytic activity of fiber degrading enzyme irrespective of fungal habitat of buffalo and cattle.