Staphylococcus aureus is a drug resistant pathogen with zoonotic potential commonly isolated from humans and animals. The aim of this study was to compare the occurrence of drug resistance, resistance genes, sequence types (STs), and genotypes of S. aureus isolated from humans, livestock, and wildlife in eastern Poland. A high percentage of isolates resistant to penicillin (63%), erythromycin (39%), clindamycin (37%), tetracycline (31%), and methicillin (MRSA-19%), an intermediate resistant to vancomycin (VISA-13%), and a multidrug resistant (MDR-39%) was obtained. Multilocus sequence typing analysis showed the presence of 35 different STs (with dominance ST 15, ST 45, ST 7, and ST 582 in human, and ST 398 and ST 8139 in porcine and cattle isolates, respectively), including 9 new ones that had never been reported before (ST 8133-8141). Identical genotypic patterns were detected among porcine and cattle isolates as well as from humans and cattle. A high percentage of MDR, MRSA, and VISA in humans and livestock combined with the presence of the same genotypes among S. aureus isolated from human and cattle indicates the circulation of strains common in the region and their zoonotic potential. There is a need to develop new strategies to counteract this phenomenon according to the One Health policy.