We performed a randomized prospective study of 5-day treatment with topical mupirocin or bacitracin for the elimination of Staphylococcus aureus nasal colonization in healthcare workers (HCWs). Nasal cultures were obtained from 141 HCWs, 37 (26%) of whom showed S aureus. After 72 to 96 hours of treatment, the organism was eradicated in 15 (94%) of 16 by mupirocin and in 8 (44%) of 18 by bacitracin (P=.0031). Similar efficacy was demonstrated at 30 days. Mupirocin may be more effective than bacitracin for eradication of S aureus in healthy HCWs.