Systemic treatment of biomaterial-associated bacterial infections with high doses of antibiotics is an established therapeutic concept. The purpose of this in vitro study was to determine the influence of magnetic, electromagnetic, and electric fields on gentamicin-based, antibiotic therapy. It has been previously reported that these fields are successful in the treatment of bone healing and reducing osteitis in infected tibia-pseudarthroses. Four separate experimental setups were used to expose bacterial cultures of Staphylococcus aureus both in Mueller-Hinton broth (MHB) and on Mueller-Hinton agar (MHA), in the presence of gentamicin, to (1) a low-frequency magnetic field (MF) 20 Hz, 5 mT; (2) a low-frequency MF combined with an additional alternating electric field (MF + EF) 20 Hz, 5 mT, 470 mV/cm; (3) a sinusoidal alternating electric field (EF AC) 20 Hz, 470 mV/cm; and (4) a direct current electric field (EF DC) 588 mV/cm. No significant difference between samples and controls was detected on MHA. However, in MHB each of the four fields applied showed a significant growth reduction of planktonically grown Staphylococcus aureus in the presence of gentamicin between 32% and 91% within 24 h of the experiment. The best results were obtained by a direct current EF, decreasing colony-forming units (CFU)/ml more than 91%. The application of electromagnetic fields in the area of implant and bone infections could offer new perspectives in antibiotic treatment and antimicrobial chemotherapy. Bioelectromagnetics 32:367–377, 2011. © 2011 Wiley-Liss, Inc.