One of the most common pathogens causing musculoskeletal infections remains Staphylococcus aureus. The aim of this multicentre study was to perform a phenotypic and genotypic characterisation of clinical S. aureus isolates recovered from musculoskeletal infections and to investigate differences between isolates cultured from Orthopaedic Implant Related Infections (OIRI) and those from Non-Implant Related Infections (NIRI). OIRI were further differentiated in two groups: Fracture Fixation-Device Infections (FFI) and Prosthetic Joint Infections (PJI). Three-hundred and five S. aureus strains were collected from 4 different Swiss and 2 French hospitals (FFI, n=112; PJI, n=105; NIRI, n=88). NIRI cases were composed of 27 Osteomyelitis (OM), 23 Diabetic Foot Infections (DFI), 27 Soft Tissue Infections (STI) and 11 postoperative Spinal Infections (SI). All isolates were tested for their ability to form biofilm, to produce staphyloxanthin and their haemolytic activity. They were typed by agr (accessory gene regulator) group, spa type and screened by PCR for the presence of genes of the most relevant virulence factors such as MSCRAMMs, Panton Valentine Leukotoxin (PVL), enterotoxins, exotoxins and toxic shock syndrome toxin. Overall, methicillin susceptible S. aureus (MSSA) was more prevalent than methicillin resistant S. aureus (MRSA) in this collection. The OIRI group trended towards a higher incidence of MRSA, gentamicin resistance and haemolysis activity than the NIRI group. Within the OIRI group, PJI isolates were more frequently strong biofilm formers than isolates from the FFI group. A statistically significant difference was observed between OIRI and NIRI isolates for the sdrE gene, the cna gene, the clfA gene and the bbp gene. Certain spa types (t230 and t041) with a specific genetic virulence pattern were only found in isolates cultured from OIRI. In conclusion, our study highlights significant trends regarding the virulence requirements displayed by S. aureus isolates associated with implant related infections in comparison to non-implant related infections. However, future studies including whole genome sequencing will be required to further examine genomic differences among the different infection cases.