Background Staphylococcus aureus is one of the prevalent etiological agents of contagious bovine mastitis, causing a significant economic burden on the global dairy industry. Given the emergence of antibiotic resistance (ABR) and possible zoonotic spillovers, S aureus from mastitic cattle pose threat to both veterinary and public health. Therefore, assessment of their ABR status and pathogenic translation in human infection models is crucial. Results In this study, 43 S. aureus isolates associated with bovine mastitis obtained from four different Canadian provinces (Alberta, Ontario, Quebec, and Atlantic provinces) were tested for ABR and virulence through phenotypic and genotypic profiling. All 43 isolates exhibited crucial virulence characteristics such as hemolysis, and biofilm formation, and six isolates from ST151, ST352, and ST8 categories showed ABR. Genes associated with ABR (tetK, tetM, aac6’, norA, norB, lmrS, blaR, blaZ, etc.), toxin production (hla, hlab, lukD, etc.), adherence (fmbA, fnbB, clfA, clfB, icaABCD, etc.), and host immune invasion (spa, sbi, cap, adsA, etc.) were identified by analyzing whole-genome sequences. Although none of the isolates possessed human adaptation genes, both groups of ABR and antibiotic-susceptible isolates demonstrated intracellular invasion, colonization, infection, and death of human intestinal epithelial cells (Caco-2), and Caenorhabditis elegans. Notably, the susceptibilities of S. aureus towards antibiotics such as streptomycin, kanamycin, and ampicillin were altered when the bacteria were internalized in Caco-2 cells and C. elegans. Meanwhile, tetracycline, chloramphenicol, and ceftiofur were comparatively more effective with ≤ 2.5 log10 reductions of intracellular S. aureus. Conclusions This study demonstrated the potential of S. aureus isolated from mastitis cows to possess virulence characteristics enabling invasion of intestinal cells thus calling for developing therapeutics capable of targeting drug-resistant intracellular pathogens for effective disease management.