Peri-implant mucositis and peri-implantitis are biofilm-related diseases causing major concern in oral implantology, requiring complex anti-infective procedures or implant removal. Microbial biosurfactants emerged as new anti-biofilm agents for coating implantable devices preserving biocompatibility. This study aimed to assess the efficacy of rhamnolipid biosurfactant R89 (R89BS) to reduce Staphylococcus aureus and Staphylococcus epidermidis biofilm formation on titanium. R89BS was physically adsorbed on titanium discs (TDs). Cytotoxicity of coated TDs was evaluated on normal lung fibroblasts (MRC5) using a lactate dehydrogenase assay. The ability of coated TDs to inhibit biofilm formation was evaluated by quantifying biofilm biomass and cell metabolic activity, at different time-points, with respect to uncoated controls. A qualitative analysis of sessile bacteria was also performed by scanning electron microscopy. R89BS-coated discs showed no cytotoxic effects. TDs coated with 4 mg/mL R89BS inhibited the biofilm biomass of S. aureus by 99%, 47% and 7% and of S. epidermidis by 54%, 29%, and 10% at 24, 48 and 72 h respectively. A significant reduction of the biofilm metabolic activity was also documented. The same coating applied on three commercial implant surfaces resulted in a biomass inhibition higher than 90% for S. aureus, and up to 78% for S. epidermidis at 24 h. R89BS-coating was effective in reducing Staphylococcus biofilm formation at the titanium implant surface. The anti-biofilm action can be obtained on several different commercially available implant surfaces, independently of their surface morphology.