This work was carried out in order to characterise physiological and functional properties of bacteria from the Lactobacillus and Bifidobacterium genera, which are present in the freeze-dried probiotic formulations. Bacterial strains were isolated from different commercially available probiotic supplements using selective media isolation. Phenotypic characterization of the isolates was performed by selection of the gram-positive, non-sporogenic, catalase-negative bacteria and analysis of fermentation profiles of Lactobacillus bacteria with standardized biochemical tests of carbohydrate metabolism and other substrates using API 50 CHL test. After the phenotypic characterization of isolated bacterial strains, the functionality of these strains is defined by analysis of the most important probiotic criteria. During exposure to simulated gastrointestinal tract conditions, the number of viable cells of all tested strains, except strains Bifidobacterium sp. 3, Bifidobacterium sp. 2B and Bifidobacterium sp. 6B was held above 106 CFU/ml, where strain Lactobacillus sp. 7A showed the best viability, which number of viable cell count decreased by only 0,94 log units. All tested strains showed good auto-aggregate properties and bacterial strains Lactobacillus sp. 7A and Lactobacillus sp. 2A co-aggregated with the largest percentage with Escherichia coli 3014 (approx 55%), Lactobacillus sp. 1, Lactobacillus sp. 8/1 i Bifidobacterium sp. 8/2 co-aggregated approx 40%, while strains Lactobacillus sp. 1, Lactobacillus sp. 2A and Lactobacillus sp. 7A co-aggregated the best with Salmonella enterica serovar Typhimurium FP1 (approx 50%) and Lactobacillus sp. 8/1 co-aggregated approx 30%. High percentage of cell adhesion of strains Bifidobacterium sp. 3, Lactobacillus sp. 8/1 and Bifidobacterium sp. 8/2 to the hexane indicates their hydrophobicity, and the affinity of all tested strains of chloroform, to the electron-donor properties of the surface of bacterial cells. All strains showed weak activity hydrolase bile salts because they are deconjugate only 10-15% taurocholate added in the culture medium.