The study was carried out on the effect of administration of anaerobic fungi Neocallimastix sp. GR1 on feed intake, growth, ruminal fermentation, rumen microbial population and nutrient digestion in female Murrah buffalo calves (5 to 8 months of age) for a period of 90 days. In fungus-fed calves (250 ml broth @ ≈10⁶ tfu/ ml; every 4^th day), a considerable increase in daily weight gains (140 g) and better feed efficiency of total mixed ration (3.61% units) were found when compared with control calves. Besides, the digestibility of dry matter, crude protein, neutral detergent fibre and acid detergent fibre were also higher (P≤0.05) in fungus-fed calves. The digestible energy of total mixed ration in terms of total digestible nutrients was also enhanced. The pH and NH₃-N of rumen liquor were found to be lowered, whereas total-N, tricarboxylic acid precipitable-N and total volatile fatty acids and number of zoospore/ml were higher. Hence, the present study suggests that rumen fungi Neocallimastix sp. could be exploited as direct-fed microbials for domesticated ruminants to improve the nutritive value of straw based diets.