The objective of this study was to assess the rate of nosocomial infections (NIs), frequency of nosocomial pathogens and antimicrobial susceptibility changes in a 530-bed hospital over a five-year period. Hospital-wide laboratory-based NI surveillance was performed prospectively between 1999 and 2003. The Centers for Disease Control and Prevention's definitions were used for NIs and nosocomial surgical site infections, and NI rates were calculated by the number of NIs per number of hospitalized patients on an annual basis. NI rates ranged between 1.4% and 2.4%. Higher rates were observed in the neurology, neurosurgery, paediatric and dermatology departments; the low rate of NIs overall may be due to the surveillance method used. The most commonly observed infections were urinary tract, surgical site and primary bloodstream infections, and the most frequently isolated pathogens were Escherichia coli, Klebsiella pneumoniae, Enterococcus spp. and Staphylococcus aureus. Carbapenems were the most effective agents against enterobacteriaceae. Meticillin resistance among S. aureus isolates was less than 50%, and all S. aureus and Enterococcus spp. isolates were susceptible to glycopeptides apart from one glycopeptide-resistant E. faecium isolate identified in 2003. Data obtained by the same method enabled comparison between years and assisted in the detection of recent changes. Antimicrobial susceptibility data on nosocomial pathogens provided valuable guidance for empirical antimicrobial therapy of NIs.