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Background The objective of this study was to assess the effects of diet on bacterial species in the solid fraction of the ruminal content using the gene sequences of the conserved 16S rDNA region steers fed one of the following diets: canola (C), cottonseed (A), sunflower (G), soybean (SO), corn silage (S) and control diet (PD). Canola, cottonseed, sunflower and soybean were fed as whole seeds. Six crossbred steers (Body weight = 416.33 ± 93.30 kg; mean ± SD), castrated male, and fitted with ruminal cannula were used. The experimental design was a 6 × 6 Latin square design. Results Cellulolytic bacteria were predominant for all diets, with 47.75% of Operational Taxonomic Units (OTU) in animals fed the cottonseed diet. Amylolytic bacteria were identified for all diets, representing 62.51% OTU in animals consuming the sunflower diet. Proteolytic bacteria were identified for all diets, corresponding to 65.96% OUT in animals fed the sunflower diet. Lactic bacteria were identified for all diets. Megasphaera elsdenii bacterium was identified for all diets, with a greater diversity of this bacterium in steers fed the control diet. This bacterium may reduce the availability of hydrogen in the rumen due to propionate production and lactate utilization. Conclusion Oilseed in the diet showed a similarity of bacteria species with 47.5% of changing of the ruminal flora.