Implant-related infections are serious complications of trauma and orthopedic surgery and are most difficult to treat. The bacterial biofilms of 34 clinical Staphylococcus sp. isolates (Staphylococcus aureus, n = 14; coagulase-negative staphylococci, n = 19) were incubated with daptomycin (DAP; 5, 25, or 100 mg/liter), vancomycin (VAN; 5, 25, or 100 mg/liter), tigecycline (TGC; 1, 5, or 25 mg/liter), fosfomycin (FOM; 100, 250, or 1,000 mg/liter), and cefamandole (FAM; 50, 100, or 500 mg/liter) for 24 h at three different ambient temperatures: 35°C, 40°C, and 45°C. To quantify the reduction of the biomass, the optical density ratio (ODr) of stained biofilms and the number of growing bacteria were determined. Increasing the temperature to 45°C or to 40°C during incubation with FAM, FOM, TGC, VAN, or DAP led to a significant but differential reduction of the thickness of the staphylococcal biofilms compared to that at 35°C (P < 0.05). Growth reduction was enhanced for DAP at 100 mg/liter at 35°C, 40°C, and 45°C (log count reductions, 4, 3.6, and 3.3, respectively; P < 0.05). A growth reduction by 2 log counts was detected for FAM at a concentration of 500 mg/liter at 40°C and 45°C (P = 0.01). FOM at 1,000 mg/liter reduced the bacterial growth by 1.2 log counts (not significant). The antibacterial activity of antimicrobial agents is significantly but differentially enhanced by increasing the ambient temperature and using high concentrations. Adjuvant hyperthermia may be of value in the treatment of biofilm-associated implant-related infections.