Over a period of 7 months, 23 patients hospitalized in a neonatal intensive care unit (NICU) developed nosocomial Enterobacter cloacae bacteraemia. Contaminated saline for preparing heparin solution was initially identified as the common source of E. cloacae bacteraemia. Although environmental sanitation was enforced, the outbreak continued. E. cloacae has always been isolated from various cultures of the environmental specimens, from the hands of personnel and from the faeces of patients. All of the 23 bacteraemic isolates and 8 stool isolates from infected infants, as well as the 17 isolates from environmental specimens were found to be of the same genotype using the polymerase chain reaction-based DNA fingerprinting method. After various infection control methods were instituted, the outbreak eventually came under control. For epidemiological investigation, 23 neonates without E. cloacae bacteraemia were matched for case-control study. Nineteen (83%) of the case-patients were premature. The significant risk factors leading to E. cloacae bacteraemia in the NICU included small gestation age, low birthweight, exposure to personnel with contaminated hands and the presence of E. cloacae in the stool carriage (p=0.003, 0.007, 0.018 and 0.040, respectively). The gastrointestinal tracts of the patients and environmental surfaces appeared to be the principal sites of bacterial reservoir. In conclusion, the outbreak of E. cloacae bacteraemia was caused by a particular strain and possibly via multiple modes of transmission, including a bottle of contaminated saline as an initial common source, endogenous spread from the gastrointestinal tract and successive cross-infections between patients, hands of personnel and the environment. Effective infection control requires a multidisciplinary approach and reinforcement of infection control procedures, including aseptic technique, hand washing, proper isolation and disinfection of environmental surfaces.