To provide a comprehensive examination of the bacterial diversity in the rumen content of cows fed different diets, high-throughput 16S rRNA gene-based pyrosequencing was used. Four rumen fistulated nonlactating Holstein cows received 12 kg of dry matter per day of four diets based on maize silage during four periods: the low-starch diet (22% starch, 3% fat); the high-starch diet, supplemented with wheat plus barley (35% starch, 3% fat); the low-starch plus oil diet, supplemented with 5% of sunflower oil (20% starch, 7.6% fat) and the high-starch plus oil diet (33% starch, 7.3% fat). Samples were taken after 12 days of adaptation, 5 h postfeeding. Whatever the diet, bacterial community of sieved rumen contents was dominated by Firmicutes and Bacteroidetes. Lachnospiraceae, Ruminococcaceae, Prevotellaceae, and Rikenellaceae families were highly present and were clearly affected by cow diet. The highest abundance of Prevotellaceae and the lowest abundance of Ruminococcaceae and Rikenellaceae were found with the high-starch plus oil diet. Dietary starch increased the relative abundance of only three genera: Barnesiella, Oribacterium and Olsenella, but decreased the relative abundances of several genera, with very significant effects for Rikenellaceae_RC9 and Butyrivibrio-Pseudobutyrivibrio. Oil alone had a limited effect, but interestingly, starch plus oil addition differently affected the bacterial populations compared to starch addition without oil.