The genus Bifidobacterium has been suggested to be capable of performing gastrointestinal modifications, providing nutritional value when added to the diet as a probiotic and having an inducing effect on the immune system by enhancing cytokine production. As a consequence, the dairy food industry is introducing bifidobacteria to such products as yogurt, flavored milk, and cottage cheese. The objectives of this project were to characterize isolates of bifidobacteria from commercial suppliers, to isolate and characterize bifidobacteria from dairy products, and to establish and compare their physiological characteristics. Physiological characterization was performed based on phenotypic characteristics, carbohydrate fermentation patterns, enzyme profiles, fructose-6-phosphate phosphoketolase (F6PPK) assays, and antibiotic sensitivities. The results demonstrated that commercial bifidobacteria strains and those strains isolated from dairy products were physiologically different. This demonstrates that some bifidobacteria present in dairy products may be mischaracterized when identifying their presence based solely on phenotypic characteristics. The difficulty in growth, rapid assessment, and isolation of bifidobacteria from a mixed culture in dairy products was evaluated in this study. X-α-Gal medium was selected as the most suitable for isolation of bifidobacteria from mixed culture products and modified lactobacilli MRS medium for growing pure isolates of bifidobacteria.