The aim of this paper was investigation of lactic acid bacteria strains L. lactis ZG5-9 and E. faecium ZG1-54, isolated from the autochthonous fresh cheese, as functional starter cultures. Autochthonous lactic acid bacteria isolates were susceptible to most antibiotics tested, and are therefore safe from the standpoint of controlling the spread of antibiotic resistance. Strain L. lactis ZG5-9 is sensitive to all 12 tested antibiotics, a strain of E. faecium ZG1-54 is resistant to streptomycin and neomycin. Both bacterial strains showed good survival in simulated gastrointestinal system. Growth of selected strains of E. faecium ZG1-54 and L. lactis ZG5-9, a change in pH and lactic acid synthesis were highest during cultivation in nutrient media with mannitol and lactulose as prebiotic substrates. The cells of E. faecium ZG1-54 and L. lactis ZG5-9 showed hydrophilic properties. Test results of microbial adhesion to chloroform show an affinity for the tested LAB to acid solvent, which leads to their electron-donor (alkaline) performance. Bacteria E. faecium ZG1-54 and L. lactis ZG5-9 assimilate cholesterol in the presence of bile salts. Those strains and strain Lactobacillus pentosus ZG2-25 were used as functional starter cultures for fresh cheese production with controlled fermentation. Mass, yield, pH value and percentage of syneresis of probiotic cheese producted with autochthonous strains of Lactococcus lactis ZG5-9, Lactobacillus pentosus ZG2-25 and Enterococcus faecium ZG1-54 and the cheese produced with commercial starter culture LL-50Y were not significantly different.