Infection is a common complication of open fractures. Systemic antibiotics often cause adverse events before eradication of infected bone occurs. The local delivery of antibiotics and the use of implants that deliver both growth factors and antimicrobials are ways to circumvent systemic toxicity while decreasing infection and to reach extremely high levels required to treat bacterial biofilms. When choosing an antibiotic for a local delivery system, one should consider the effect that the antibiotic has on cell viability and osteogenic activity. To address this concern, osteoblasts were treated with 21 different antibiotics over 8 concentrations from 0 to 5,000 mg/ml. Osteoblast deoxyribonucleic acid content and alkaline phosphatase activity (ALP) were measured to determine cell number and osteogenic activity, respectively. Antibiotics that caused the greatest decrement include rifampin, minocycline, doxycycline, nafcillin, penicillin, ciprofloxacin, colistin methanesulfonate, and gentamicin; their cell number and ALP were significantly less than control at drug concentrations 200 mg/ ml. Conversely, amikacin, tobramycin, and vancomycin were the least cytotoxic and did not appreciably affect cell number and ALP until very high concentrations were used. This comprehensive evaluation of numerous antibiotics' effects on osteoblast viability and activity will enable clinicians and researchers to choose the optimal antibiotic for treatment of infection and maintenance of healthy host bone. ß