Staphylococcal-associated surgical site infections (SSI) are common nosocomial infections in healthcare facilities worldwide. The use of antiseptic-coated sutures has been recommended to minimise the risk of SSI in clinical settings. However, as there has been a growing concern over antibiotic resistance resulting from antiseptic usage, development of antimicrobial sutures using alternative compounds is necessary. In this study, menadione (2-methyl-1,4-napthoquinone), also known as Vitamin K3, was evaluated as a potential antimicrobial compound for suture coating. The anti-staphylococcal activity of menadione was assessed using microbroth dilution method and biofilm inhibition assays. The low menadione minimum biofilm inhibitory concentration values against both methicillin-susceptible and -resistant S. aureus strains indicate its inhibitory activity against staphylococcal biofilm. Menadione-coated sutures were prepared by dip-coating surgical sutures in slurries containing poly(D,L-lactide-co-glycolide) polymers (either 65:35 or 75:25) and calcium stearate. Zone of inhibition assays showed dose-dependent antimicrobial effects of the sutures up to four days. A ∼3 log10 colony forming unit/ml reduction of adherent bacteria (p < 0.05) on the sutures was demonstrated via bacterial adherence assays. The integrity and tensile strength of the sutures were unaffected by the coating procedure. In view of the increased antibiotic resistance and limited antimicrobials, menadione may be potentially useful for antimicrobial coating of surgical sutures.