The aim of this study was to identify beneficial bacteria with probiotic potential from kefir grains. The lactobacilli isolated from kefir grains were characterised as: Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus paracasei, and Lactobacillus kefiri. The strains Lb. plantarum 1Ž, Lb. paraplantarum S10, and Lb. paracasei 2Ž tolerated better the test gastric juice at pH 2 and 2.6 during 120 min of incubation in comparison with the strains Lb. kefiri. On the other hand, the strains Lb. kefiri were resistant to 0.3 % bile acid salts. The Lb. paracasei 2Ž showed the significantly highest survival (P < 0.001) at pH 2 in comparison with all other strains tested and was also able to tolerate 0.3 % concentration of the bile salts. All strains produced medium to strong biofilms on abiotic surfaces and inhibited the growth of selected potential pathogens with varying intensity. All kefir isolates were susceptible to the antibiotics tested and exhibited positive β-galactosidase activity with the exception of Lb. paracasei 2Ž which did not show any activity of undesirable enzymes, such as β-glucosidase and β-glucuronidase. Additional testing and validation of the biological properties and safety of the strain Lb. paracasei 2Ž under in vivo conditions are needed to confirm the prospective use of this strain in practice.