Coagulase-negative staphylococci are important nosocomial pathogens that increasingly are resistant to oxacillin and fluoroquinolones. To determine predictors of acquisition of oxacillin and ofloxacin resistance, we prospectively identified 150 patients from whose clinical specimens coagulase-negative staphylococci were isolated that differed in susceptibility to oxacillin and ofloxacin. In multivariate analysis, isolation of ofloxacin-resistant coagulase-negative staphylococci was associated with receipt of aminoglycosides (odds ratio [OR] = 8.45; 95% confidence interval [CI] = 2.10-34.1; P = .001) and fluoroquinolones (OR = 11.50; 95% CI = 4.15-31.6; P < .001) within 30 days; oxacillin resistance was associated with prior receipt of beta-lactam agents (OR = 5.99; 95% CI = 2.91-12.3; P < .001). Among oxacillin-resistant strains, there was heterogeneity of pulsed-field gel electrophoresis (PFGE) types, and no type was common between ofloxacin-resistant and ofloxacin-susceptible strains. Thus ofloxacin resistance may have emerged de novo among diverse oxacillin-resistant strains following the selection pressures of antimicrobial therapy. In contrast, 50% of patients with oxacillin-susceptible/ofloxacin-resistant strains had one of two PFGE types, a finding suggesting that person-to-person transmission resulted in the dissemination of some of these strains.