Water buffalo produce a tenth of milk for global human consumption. Non-aureus staphylococci (NAS) are among the most commonly isolated bacteria from mastitis in water buffalo and dairy cows. These results described the initial characterisation of 17 NAS-15 Staphylococcus simulans and two Staphylococcus chromogenes from a water buffalo herd (n = 44) in South Africa. The isolates were identified by classical microbiology, MALDI-TOF, and 16S rRNA, and the disc diffusion method determined the antibiotic susceptibility. A multi-locus sequence typing scheme (MLST) was developed to determine S. simulans sequence types (ST), by defining and comparing seven housekeeping gene fragment sequences. Sequence typing confirmed all 15 S. simulans isolates from water buffalo which belonged to a single ST, genetically distant from the six bovine STs isolated from adjacent farms, which also varied, indicating no current bacterial transfer between species. The antibiotic resistance patterns of S. simulans varied between beta-lactams. The mean milk somatic cell count (SCC) for the water buffalo milk samples was 166,500 cells/mL milk. This information offers insights into the epidemiology and comparison among isolates from various origins, which leads to effective proactive mastitis strategies resulting in safe, high-quality dairy products from water buffalo and dairy cows for human consumption.