We conducted a retrospective analysis of all bile specimens obtained for routine cultures from January 1995 through December 1999 at our tertiary care hospital. Results of microbiologic testing were linked to clinical parameters gathered by means of chart review. A total of 722 isolates were cultured from 345 of 454 bile specimens obtained from 288 individual patients. Prior receipt of a >7-day course of antibiotics (odds ratio [OR], 5.7), extensive leukocytosis (leukocyte count, >20,000 cells/microL) on admission (OR, 7.8), endoscopic or percutaneous biliary manipulation during the previous 14 days (OR, 2.9), and treatment in an internal medicine ward (OR, 2.5) were independent factors significantly associated (Pless-than-or-eq, slant.05) with recovery of Candida species from bile specimens. Culture of mezlocillin-resistant bacteria from bile specimens was independently associated with the specimen having been obtained >1 week after admission (OR, 3.8), lack of history of endoscopic biliary drainage (OR, 3.2), and high serum aspartate aminotransferase levels (>72 U/L) on admission (OR, 2.6). Prospective studies are warranted to evaluate accordingly adjusted empiric therapies for biliary infections.