BACKGROUND: Identifying the potential of livestock and forage plants of each region is a way to compensate the lack of forage, and efficient use of edible resources of the country. Milk Thistle is a medicinal plant which may be used as fodder for native livestock of Khuzestan Province. OBJECTIVES: The purpose of this research was to study  the effects of different levels of Milk Thistle on rumen fungi and whole rumen microorganisms (WRM) of Khuzestan buffalo, with diets containing grains with different degradation rate (barley and maize). METHODS: The impact of diets containing different levels of Milk Thistle (0, 100 and 200 g per kg DM) on rumen fungi and WRM of buffalo was measured by different techniques. RESULTS: Potential and rate of gas production from experimental diets by WRM were not significantly different. This parameters by buffalo rumen fungi were significantly differed and increased in diets containing Milk Thistle (p<0.05). Adding Milk Thistle in the barley-based diet increased dry matter and NDF digestibility numerically while in the corn-based diet dry matter and NDF digestibility was slightly reduced. In the SRFCM, NDF digestibility, in both basal diets in 9 days and during total period was affected by diets containing Milk Thistle and decreased NDF digestibility with increasing levels of Milk Thistle (p<0.05). Regardless of the type of basal diet, digestibility of NDF (p<0.05) and dry matter (p>0.05) for day 6, were increased with increasing the amount of Milk Thistle. CONCLUSIONS: In general, the use of Milk Thistle did not have negative effect on microorganisms and digestion of nutrients by them. Therefore, results suggest that Milk Thistle could be used up to 20 percentage in buffalo diet without any negative effect on digestion and fermentation characteristics by total microorganisms and fungi.