Biofilms have an important role in the pathogenesis of Periprosthetic Joint Infection (PJI)s and are very likely the main cause in resistance to conventional treatments. In this study, a novel thermosensitive, Pluronic - based gel formulation, containing an enzyme (Serratiopeptidase, SP), an antibiotic drug (Vancomycin HCl, VA) and antibiotic-loaded microspheres (TG-EAM) were prepared and evaluated for their effect against PJI. Viscosity measurements, drug and enzyme release, in vitro enzyme activity and in vitro anti-biofilm activity studies were performed to characterize the formulations. While in vitro drug release studies showed that 74.5% VA was released after 25 d from TG-EAM, the in vitro enzyme activity was measurable for two days. Based on the obtained experimental results, the formulation containing 0.8% (w/v) free VA, 5% (w/v) SP and 40% (w/v) of VA loaded microspheres was chosen as the most suitable formulation for future in vivo studies, as a promising alternative to current standard treatments for PJI.