The main objective of this research was to investigate the antibiotic susceptibility of autochthonous lactic acid bacteria (LAB) isolated from traditional Croatian fresh cheese which are selected as functional starter cultures for fresh cheese production under controlled industrial conditions. Soluble whole cell protein patterns of dominant autochthonous LAB strains from fresh cheese, obtained by SDS-PAGE, actually revealed the presence of two different strains, which were identified as Lactobacillus fermentum A8 and Enterococcus faecium A7. Based on agar disc diffusion test and E-test, antibiotic susceptibility as well as presence of plasmids in autochthonous starter cultures Lb. fermentum A8 and Ec. faecium A7 were evaluated. Resistance to commonly used antibiotics of different classes was assessed by disk diffusion method. The study also included determination of MICs for both strains by E-test. Lb. fermentum A8 and Ec. faecium A7 were susceptible to tetracycline, gentamicin and erythromycin, three clinically most commonly applied antibiotics. In addition, tested strains were also susceptible to inhibitors of cell-wall synthesis: ampicillin, bacitracin, penicillin G, rifampicin; inhibitors of protein synthesis: azithromycin, chloramphenicol, clarithromycin, clindamycin, spiramycin and to inhibitors of DNA synthesis: rifampicin and novobiocin. Lb. fermentum A8 strain displayed phenotypic resistance to vancomycin, but this resistance is intrinsic and not transferable. According to obtained results Lb. fermentum A8 and Ec. faecium A7 are functional starter cultures that are safe from the aspect of spreading antibiotic resistance through cheese as food matrix and could be applied as efficient starter cultures for fresh cheese production, that show antimicrobial activity against Escherichia coli 3014 and Salmonella enterica serovar Typhimurium FP1.