BACKGROUND: Acinetobacter spp. is an important nosocomial pathogen for which increasing resistance to multiple antimicrobial agents has been observed. Prevalence of multidrug-resistant (MDR) Acinetobacter spp. in the intensive care unit (ICU) at a teaching hospital in Korea started to increase in 2008. The aim of this study was to determine the source of pathogen spread and to characterize the emerging strains at an early stage of outbreak. METHODS: Samples from respiratory instruments and fomites in the ICUs, as well as from the healthcare workers, were cultured to identify the sources of MDR Acinetobacter spp. Antimicrobial susceptibility was determined by the CLSI disk diffusion method. Pulsed field gel electrophoresis (PFGE) was performed for clinical and environmental isolates in order to determine clonality. Carbapenemase genes were detected by multiplex PCR. Infection control measures including peer-monitoring of hand washing, environmental cleaning and standard precautions were enforced. RESULTS: Among the samples from the ICU tools (105) and healthcare worker's hands (44), 31 (30%) and 2 (5%) respective samples yielded MDR Acinetobacter spp. Among the environmental samples, 90% were from respiratory-related equipment. The majority of clinical and environmental MDR Acinetobacter spp. (44/55) belonged to the pulsotype A. baumannii and carried both bla(OXA-51)-like and bla(OXA-23)-like genes. Even though infection-control measures were enforced, prevalence of MDR Acinetobacter spp. continues to increase. CONCLUSION: An outbreak of MDR Acinetobacter spp. in a Korean hospital was caused by A. baumannii carrying the bla(OXA-23)-gene and was correlated with contaminated respiratory-related instruments in the ICUs. More intensive measures for nosocomial infection control are needed for successful prevention of Acinetobacter spread in hospitals.