Staphylococcus aureusis a common cause of infection in humans and animals, including bovine mastitis, globally. The objective of this study was to genetically characterize a collection ofS. aureusisolates recovered from milk and nasal swabs from humans with and without animal contact (bovine = 43, human = 12). Using whole genome sequencing (NextSeq550), isolates were sequence typed, screened for antimicrobial resistance and virulence genes and examined for possible inter-species host transmission. Multi locus sequence typing (MLST) and single nucleotide polymorphism (SNP)-based phylogeny revealed 14 different sequence types, including the following six novel sequence types: ST7840, 7841, 7845, 7846, 7847, and 7848. The SNP tree confirmed that MLST clustering occurred most commonly within CC97, CC5477, and CC152. ResFinder analysis revealed five common antibiotic resistance genes, namelytet(K),blaZ,dfrG,erm©, andstr, encoding for different antibiotics.mecAwas discovered in one human isolate only. Multidrug resistance was observed in 25% of the isolates, predominantly in CC152 (7/8) and CC121 (3/4). Known bovineS. aureus(CC97) were collected in humans and known humanS. aureuslineages (CC152) were collected in cattle; additionally, when these were compared to bovine-isolated CC97 and human-isolated CC152, respectively, no genetic distinction could be observed. This is suggestive of inter-host transmission and supports the need for surveillance of the human–animal interface.