Sorghum presents a sustainable feedstock for Mediterranean buffaloes due to its reduced water and nitrogen requirements compared with maize, which is currently fed primarily. We investigated the effects of feeding sorghum as opposed to maize on Mediterranean buffalo rumen microbial diversity and milk fatty acid content. Four cannulated lactating Mediterranean buffalo cows were fed a basal diet for one month before switching either to maize or sorghum-silage based diets for a 3-month period. Buffaloes were then changed over to the contrasting diet for a further one month. Rumen and milk samples were collected at the end of each month. DGGE- and T-RFLP-based dendrograms generated from rumen samples did not show an effect of diet on rumen bacterial diversity. Milk samples also did not differ in terms of their fatty acid content post sorghum feeding as compared with maize feeding. Thus, sorghum provides an environmentally beneficial alternative to maize for feeding Mediterranean buffalo with little effect on rumen microbial diversity or milk fatty acid composition compared with maize feeding.