Aims: To examine the diversity of Staphylococcus aureus isolated from nasal swabs of ruminants in Rwanda. Methods and results: A total of 454 nasal swabs from 203 cows, 170 goats, and 81 sheep were examined for the presence of S. aureus, and 30 S. aureus isolates were detected and characterized pheno- and genotypically. Resistance to penicillin and/or tetracycline was observed. The isolates were assigned to eight different spa types (t21057 (novel), t10103, t18853, t20842, t318, t355, t458, and t9432) belonging to six clonal complexes (CCs) (CC152, CC30, CC3591, CC3666, CC522, and CC97). Panton–Valentine leukocidin (PVL) genes (lukF-PV/lukS-PV), the bovine leukocidin genes (lukM/lukF-P83), and the human and bovine variants of the toxic shock syndrome toxin gene tst-1 variants were detected. Conclusion: These findings demonstrate that the nares of ruminants in Rwanda are colonized with mastitis-associated S. aureus, including lineages that are also carried by humans, underscoring the zoonotic risk, especially for livestock keepers. These results highlight the crucial importance of hygiene measures when handling livestock. Impact Statement This study significantly improved our knowledge on the ruminant-associated Staphylococcus aureus in Rwanda. Various virulence-associated genes, including Panton–Valentine leukocidin and the tst-1 genes were detected suggesting that ruminants carry S. aureus strains potentially pathogenic for humans.