The widespread use of antimicrobial substances has led to resistant populations of microorganisms in several ecosystems. In animal husbandry, the application of antibiotics has contributed to resistance development in pathogenic and commensal bacteria. These strains or their resistance genes can be spread along several ecological routes, including the food chain. Antibiotic resistance is important in terms of the safety of industrial strains, such as probiotics for food and feed. Bifidobacterium thermophilum and Bifidobacterium pseudolongum are known to comprise the major part of the bifidobacterial microbiota in the gut and feces of cattle and pigs. In this study, the antimicrobial susceptibility in bifidobacterial isolates of these species was investigated. Isolates from the beef and pork production chain were identified and typed to strain level, and the antimicrobial susceptibility level was tested to a set of antibiotics. Isolates with low susceptibility levels were screened by PCR for already described resistance genes. Strains atypically resistant to clindamycin, erythromycin, and tetracycline were determined. The resistance genes tet(O), tet(W), and erm(X) were detected in the bifidobacterial species that were examined.