Abstract Staphylococcus aureus is one of the most prevalent pathogens causing bovine mastitis in the world, in part because of its ease of adaptation to various hosts and the environment. This study aimed to determine the prevalence of S. aureus in Colombian dairy farms and its relationship with the causal network of subclinical mastitis. From thirteen dairy farms enrolled, 1288 quarter milk samples (QMS) and 330 teat samples were taken from cows with positive (70.1%) and negative California Mastitis Test (CMT). In addition, 126 samples from the milking parlor environment and 40 from workers (nasal) were collected. On each dairy farm, a survey was conducted, and the milking process was monitored on the day of sampling. S. aureus was identified in 176 samples, i.e., 138 QMS, 20 from teats, 8 from the milking parlor environment, and 10 from workers’ nasal swabs. Isolates identified as S. aureus underwent proteomics (clustering of mass spectrum) and molecular ( tuf , coa , spa Ig , clfA , and eno genes) analysis. Regarding proteomics results, isolates were distributed into three clusters, each with members from all sources and all farms. Concerning molecular analysis, the virulence-related genes clfA and eno were identified in 41.3% and 37.8% of S. aureus isolates, respectively. We provide evidence on the circulation of S. aureus strains with limited variability among animals, humans, and the environment. The parameters with the lowest compliance in the farms which may be implicated in the transmission of S. aureus are the lack of handwashing and abnormal milk handling.