Subtherapeutic antibiotic use in ruminant feeding to optimize rumen fermentation may lead to residues in meat and milk (6), as well as an increase in the inhibition of ruminal bacterial populations (7). For this reason, in 2006, the European Union banned the use of antibiotics as growth promoters in livestock feeding due to potential toxicities to host animals, potential rumen microbial adaptation, and risk of the presence of residues of these compounds in milk and meat, with potential effects on human health (8).In the present study, sensitive and mutant colonies of some ruminal bacterial species isolated from sheep, cattle, and buffalo were detected. We counted and considered “mutant colonies” the bacterial colonies grown in the clear inhibition zone in the Kirby–Bauer disk diffusion susceptibility test. Detected mutant colonies were higher in buffalo than in cattle and sheep. Duricef and metronidazole caused no mutations in any species. The others formed mutant colonies, where roxithromycin = polymyxin = chloramphenicol = gentamicin < erythromycin < vancomycin < piperacillin = cefotaxime < streptomycin < cefoperazone < ciprofloxacin < amikacin. Sheep had the highest number of sensitive isolates, and the number of sensitive isolates was dramatically lower in cattle and buffalo. There were no sensitive isolates with the antibiotic metronidazole, and there was a low number of sensitive isolates with duricef. The other antibiotics had more sensitive isolates (gentamicin = ciprofloxacin = amikacin > streptomycin = piperacillin > erythromycin > vancomycin = cefoperazone = cefotaxime > roxithromycin > polymyxin > chloramphenicol). The number of sensitive isolates of the different ruminant species for all the antibiotics was highest in buffalo, followed by cattle and then sheep (P < 0.05). We could conclude that subtherapeutic antibiotic use in ruminant feeding may lead to the formation of antibiotic-resistant mutant colonies, making their subtherapeutic effect nonexistent.