Prosthetic joint infections (PJI) are frequent complications of arthroplasties. Their treatment is made complex by the rapid formation of bacterial biofilms, limiting the effectiveness of antibiotic therapy. In this study, we explore the effect of a tri-enzymatic cocktail (TEC) consisting of an endo-1,4-β-d-glucanase, a β-1,6-hexosaminidase, and an RNA/DNA nonspecific endonuclease combined with antibiotics of different classes against biofilms of Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli grown on Ti-6Al-4V substrates. Biofilms were grown in Trypticase soy broth (TSB) with 10 g/liter glucose and 20 g/liter NaCl (TGN). Mature biofilms were assigned to a control group or treated with the TEC for 30 min and then either analyzed or reincubated for 24 h in TGN or TGN with antibiotics. The cytotoxicity of the TEC was assayed against MG-63 osteoblasts, primary murine fibroblasts, and J-774 macrophages using the lactate dehydrogenase (LDH) release test. The TEC dispersed 80.3 to 95.2% of the biofilms\' biomass after 30 min. The reincubation of the treated biofilms with antibiotics resulted in a synergistic reduction of the total culturable bacterial count (CFU) compared to that of biofilms treated with antibiotics alone in the three tested species (additional reduction from 2 to more than 3 log10 CFU). No toxicity of the TEC was observed against the tested cell lines after 24 h of incubation. The combination of pretreatment with TEC followed by 24 h of incubation with antibiotics had a synergistic effect against biofilms of S. aureus, S. epidermidis, and E. coli Further studies should assess the potential of the TEC as an adjuvant therapy in in vivo models of PJI.