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Background and Aim: Enterococcus species are opportunistic pathogens and their pathogenicity seems to be related to the presence of a number of pathogenicity genes. Since donkey’s milk is a new non-allergenic source of nutrition, this study was performed to assess the antibiogram and detection of pathogenicity genes in some Enterococcus faecalis isolates from donkey’s milk. Materials and Method: In this experimental study, several Enterococcus faecalis strains were isolated from donkey’s milk. Resistance patterns of the isolates to 10 antibiotics including vancomycin were investigated based on CLSI protocol. Statistical comparison was made by Fisherchr('39')s exact test. Polymerase chain reaction (PCR) amplification was used to study gel E, esp, ace, as and efaA pathogenicity related genes. Results: Enterococcus faecalis isolates showed a different antibiogram pattern. Isolates were resistant to azithromycin, and erythromycin, but were susceptible to ampicillin and penicillin. Previously isolated LUB93929 and LUB93101 isolates were found to be susceptible and resistance to vancomycin, respectively. GelE, ace and efaA genes were detected in both Enterococcus faecalis isolates and also in E. faecalis in the control strains. The aggregation substance gene (as) was only amplified in LUB93101 isolate. Interestingly, esp gene was not detected in any of the isolates. Conclusion: Despite resistance to vancomycin and presence of some pathogenicity related genes in this study, E. faecalis isolates may not be human pathogens due to lack of pathogenic factors. The esp gene is crucial for biofilm formation and rise of nosocomial infections. Donkey’s milk Enterococcus faecalis isolates are not able to form biofilm and seem not to bring any problem.