Endogenous infections with multi-resistant S. epidermidis are among the leading causes of nosocomial infections. The effect of hospitalization and antimicrobial therapy on antimicrobial resistance of colonizing staphylococci was determined from swabs of the nose, hand, axilla and groin from 157 patients on one day. Hospitalization for >72 hours, compared with <72 hours, was associated with a higher percentage of isolates resistant to oxacillin (56% versus 19%), gentamicin (40% versus 15%), trimethoprim (36% versus 17%), clindamycin (56% versus 17%), and fusidic acid (20% versus 4%; p < 0.01 for all), but not to rifampicin (6% versus 1%) or fosfomycin (43% versus 34%, p > 0.05 for both). Concurrent antimicrobial therapy resulted in increased resistance to oxacillin (61% versus 28%), gentamicin (43% versus 20%), and clindamycin (60% versus 26%; p < 0.01 for all), but not to trimethoprim (39% versus 23%), fusidic acid (19% versus 9%), rifampicin (6% versus 3%), or fosfomycin (46% versus 38%, p > 0.05 for all). The increase in resistant isolates was not independent, since hospitalization and antimicrobial therapy were correlated (p < 0.001). After adjustment for potential risk factors such as diabetes mellitus, central venous catheters, and hemodialysis, the odds ratio for oxacillin resistance was 2.8-3.6. None of the risk factors showed statistically significant results, except for the presence of neoplastic disease, which had a significant interaction (P=0.035). The within-subgroup odds ratios for patients with and without neoplasm were 4.2 (95% CI, 2.3-5.7) and 2.1 (95% CI, 0.78-3.12), respectively. These results show that hospitalization for more than three days, with or without antimicrobial therapy, and the presence of neoplastic disease are associated with increased antimicrobial resistance in colonizing S. epidermidis.