Specific strains of Enterococcus species are used in the food industry in food fermentation and as probiotics. However, given that certain strains are opportunistic pathogens, a detailed characterization of the safety of application is required. In this paper, it is priority to define the phenotypic characteristics of Enterococcus faecium, Enterococcus faecalis and Enterococcus durans strains, related to safety of use as starter or probiotic cultures, which were previously isolated from the microbiome of autochthonous Croatian cheeses. Hemolytic and gelatinous activity, the possibility of biofilm formation, the presence of genes for enterocin A and enterocin B were defined. From a technological point of view, the lyophilization procedure of pure cultures of Enterococcus strains was carried out, in order to determine the percentage of survival during the preparation of the lyophilizate. It was found that none of the analyzed strains exhibits hemolytic or gelatinous activity. 55 % of the strains have the ability to form biofilms, which, along with the synthesis of enterocin A and/or enterocin B, indicates that certain strains have a potential application as starter cultures. All analyzed strains survive lyophilization in skimmed milk well, except for Enterococcus faecium/durans ZG4-24.