Three autochthonous strains of lactic acid bacteria, Lactococcus lactis ZG5-9, Lactobacillus pentosus ZG2-25 and Enterococcus faecium ZG1-54, isolated from traditionally produced fresh cheese were chosen to be used for the controlled production of fresh cheese with sensory characteristics similar to the traditional cheese. Bacterial strains Lactobacillus pentosus ZG225 and Lactococcus lactis ZG5-9 have been previously investigated strains within the probiotic concept. Those autochthonous lactic acid bacteria isolates are susceptible to most tested antibiotics, and are therefore safe from the standpoint of controlling the spread of antibiotic resistance. There is resistance to vancomycin, but it is the innate resistance of Lactobacillus species, and genes that are responsible are found in genomic DNA without the danger of the gene transfer. Selected autochthonous strains Lactobacillus pentosus ZG2-25 and Lactococcus lactis ZG5-9 survive simulated gastrointestinal conditions, assimilate prebiotic substrates inulin, mannitol, lactulose and raftilozu. Growth of selected strains, changes in pH values and concentrations of lactic acid were highest during cultivation in nutrient media with mannitol and lactulose. Bakterial strains Lactobacillus pentosus ZG2-25 and L. lactis ZG5-9 assimilate cholesterol in the presence of bile salts. The cells of Lactobacillus pentosus ZG2-25 and L. lactis ZG5-9 showed hydrophilic properties and affinity for chloroform, which suggests an electron-donor (alkaline) properties of the surface of their cells. Both strains and strain of Enterococcus faecium ZG1-54 showed a very high survival rate after lyophilization process in skimmed milk as lioprotector and were used as functional starter cultures for fresh cheese production with controlled fermentation. Cheese produced with autochthonous probiotic strains was according to the sensoric properties much similar to the traditionaly produced fresh cheese then the cheese produced with commercial starter cultures.