With the aim to select new candidates for further potential use as starter or probiotic cultures, isolates from Slovak ovine and caprine cheeses were analysed by in vitro subtractive screening for safety attributes and probiotic potential using methods recommended by EFSA.The twelve strains were identified using matrix-assisted laser desorption ionization-time of flight spectrometry (Maldi-Tof MS) as Lactobacillus plantarum with score higher than 2.3 indicating highly probably identification at the species level. For antibiotic susceptibility testing, microdilution method according the ISO 10932/IDF 223 standard was used. Two strains 19l5 and 18l4 with non wild-type ECOFFs for gentamicin (>64mg/L) and/or clindamycin (>2 mg/L) were excluded. Remaining isolates expressed high bile tolerance at 0.3% oxgall after 4 h exposure (viability reached 89-99%) and survival at pH 2.5 ranged between 74-91%. From examined strains three (17l1, 18l2 and 16l4) showed no harmful β-glucuronidase or β-glucosidase activity associated with detrimental effects in the colon, and had α-fucosidase activity, which helps long-term intestinal colonization. Two strains (18l2, 16l4) exhibited β-galactosidase activity associated with positive impact in the alleviation of lactose intolerance. The three Lactobacilli without harmful properties and together strong biofilm producers were preliminary screened by an agar well diffusion assay for capacity to inhibit representative Gram-negative bacteria, such as E. coli DH5a/pCIB10B and Gram-positive bacteria as S. sciuri. For this purpose untreated acid cell free culture supernatants (ACFCSs), trypsin and proteinase K treated and neutralized supernatants prepared from the mentioned strains were used. Only ACFCSs showed antimicrobial activity and were therefore applied for viability assessment using Flow Cytometry. Survival of pathogens was measured after 24 hours of exposure with 10 % ACFCSs. The results showed that ACFCS from 18L2 isolate had strong inhibitory effect on pathogens. The percentage of live S. sciuri decreased from origin 53,57% to 0,67% and number of viable E. coli DH5a/pCIB10B from 50.88% to 2,45%. Other two isolates had significantly lower or no effect on pathogens viability.The results of subtractive in vitro screening indicate that above mentioned isolate fulfils the basic safety and functional requirements including wild-type ECOFF, lack of undesirable enzymatic activity, high acid and bile tolerance. Simultaneously 18L2 isolate exhibited β- galactosidase, α-fucosidase, produced strong biofilm and had marked inhibitory effect on viability of selected pathogens. These characteristics designate 18L2 for further investigation in in vivo studies to confirm its potential health benefits and its application as novel probiotic strains in the feed supplements.