This study was performed to evaluate the prescribing pattern of antibiotics and the relationship between antibiotic use and antibiotic resistance in major nosocomial pathogens at a university hospital in Korea. Data on annual patient-days and annual consumption (defined daily dose (DDD)/1,000 patient-days) of all antibiotics from 2000 to 2006 were analyzed. The antibiotic resistance in major nosocomial pathogens was determined by disc diffusion or automated systems. Spearman's correlation coefficient was used to determine the relationship between antibiotic use and resistance. Increasing trend on incidences of Klebsiella pneumoniae causing nosocomial bloodstream infections was noted from 2000 to 2006 (r=0.786, p=0.036). Even though the annual patient-days of the hospital did not change (r=−0.107, p=0.819), the annual usage of antibiotics significantly increased from 2000 to 2006 (r=0.929, p=0.003). A significant stepwise increase in consumption (r>0.72 and p<0.05) was found for several broad-spectrum antibiotics such as ceftriaxone, piperacillin/tazobactam, imipenem, and vancomycin. The rise in piperacillin/tazobactam-resistant Serratia marcenscens (r=0.893, p=0.007) and piperacillin/tazobactam-resistant Pseudomonas aeruginosa (r=1.000, p=0.000) was significantly correlated with increased consumption of piperacillin/tazobactam respectively. Increased ciprofloxacin-resistant S. marcescens was significantly associated with increased usage of ciprofloxacin (r=0.919, p=0.003). This study demonstrated the significant changes in antibiotic use and positive relationship between antibiotic use and antibiotic resistance in several nosocomial pathogens. Continuous surveillance of antibiotic use is needed to encourage appropriate use of antibiotics and to reduce antibiotics resistance.