Bacterial strains from the Lactobacillus and Bifidobacterium genus are most commonly characterized as probiotics. An uncontrolled use of antibiotics has influenced on the spreading of antibiotic resistance genes, which is why probiotic strains are tested for antibiotic susceptibility as one of the general selection criteria inside a strictly defined probiotic concept. In this work the antibiotic susceptibility of the probiotic strains belonging to Lactobacillus and Bifidobacterium genera, present in lyophilized probiotic preparations, are tested. A method for determining a possible presence of phenotypic resistance was the agar diffusion method with filter disks, and for determining the minimum inhibitory concentration of the antibiotic, the E-test was used. All examined strains were susceptible to ampicillin, bacitracin, chloramphenicol, clindamycin, erythromycin, gentamicin, novobiocin and penicillin G. The tested strains were also susceptible to antibiotics and inhibitors of cell wall synthesis: ampicillin, rifampicin, streptomycin; protein inhibitors: azithromycin, chloramphenicol, clarithromycin, clindamycin, spiramycin and the inhibitors DNA: rifampin and novobiocin. It has been noted that vancomycin does not have an inhibitory effect on the tested probiotic strains from Lactobacillus, which is a result of a regulated type of resistance that can be found in representative strains of this genus, and is not transferrable to distant bacterial species. Based on these results, it can be concluded that probiotic strains are safe in terms of spreading antibiotic resistance and can be applied as lyophilized probiotic formulations.