Background Central venous catheters are important in the management of paediatric intensive care unit patients, but can have serious complications which worsen the patients' health, prolong hospital stays and increase the cost of care. Evidence-based recommendations for preventing catheter-related bloodstream infections are available, but it is unknown how widely these are known or practiced in the paediatric intensive care environment. Objectives To assess nursing knowledge of evidence based guidelines to prevent catheter-related bloodstream infections; the extent to which Australia and New Zealand paediatric intensive cares have adopted prevention practices; and to identify the factors that encouraged their adoption and improve nursing knowledge. Design Cross-sectional surveys using convenience sampling. Settings Tertiary level paediatric intensive care units in Australia and New Zealand. Participants Paediatric intensive care nursing staff and nurse managers. Methods Between 2010 and 2011, the 'Paediatric Intensive Care Nurses' Knowledge of Evidence-Based Catheter-Related Bloodstream Infection Prevention Questionnaire' was distributed to paediatric intensive care nursing staff and the 'Catheter-Related Bloodstream Infection Prevention Practices Survey' was distributed to nurse managers to measure knowledge, practices and culture. Results The questionnaires were completed by 253 paediatric intensive care nurses (response rate: 34%). The mean total knowledge score was 5.5 (SD = 1.4) out of a possible ten, with significant variation of total scores between paediatric intensive care sites (p = 0.01). Other demographic characteristics were not significantly associated with variation in total knowledge scores. All nursing managers from Australian and New Zealand paediatric intensive care units participated in the survey (n = 8; response rate: 100%). Wide practice variation was reported, with inconsistent adherence to recommendations. Safety culture was not significantly associated with mean knowledge scores per site. Conclusions This study has identified that there is variation in the infection prevention approach and nurses' knowledge about catheter-related bloodstream infection prevention. The presence of an improved safety culture, years of paediatric intensive care experience and higher qualifications did not influence the nurses' uptake of recommendations, therefore further factors need to be explored in order to improve understanding and implementation of best practice.