Dietary protein plays a major role in ruminant nutrition, and protein supplementation is a widespread practice among farmers in the tropics. Ruminal bacteria are the main agents of dietary protein and amino acid degradation, yet few studies have focused on the isolation and characterization of hyper-ammonia-producing bacteria in animals fed tropical diets or supplemented with rumen-degradable proteins. This work investigated the bacterial community diversity of the rumen of Nellore steers fed tropical forages, with or without casein supplementation. We also isolated and characterized ruminal bacteria showing high levels of ammonia production. Polymerase chain reaction–denaturing gradient gel electrophoresis analysis indicated no differences in the ruminal bacterial community composition between the control and supplemented animals. Amino acid-fermenting bacteria (n = 250) were isolated from crossbred Nellore steers fed Tifton 85 (Cynodon sp.) using trypticase as the sole carbon and organic nitrogen source in the enrichment and isolation media. The deamination rates in isolates obtained from steers supplemented with casein showed a higher incidence of deamination rates >350 nmol NH3 mg protein−1 min−1 (P < 0.05), whereas isolates obtained from steers without supplementation showed deamination rates <200 nmol NH3 mg protein−1 min−1. Although most isolates (84%) could ferment carbohydrates, none could hydrolyze proteins or use urea to sustain growth. All isolates were sensitive to lasalocid and monensin (1 μmol l−1), and similarity analysis of the 16S rRNA sequences indicated a predominance of bacteria from the order Clostridiales, with variable homology (73–99%) to known bacterial species. These results expand what is known about the biochemical and genetic diversity of hyper-ammonia-producing bacteria, and emphasize the role of carbohydrate-fermenting bacteria in ammonia production in the rumen.