Background: Inappropriate needleless connector (NC) care is associated with device failure from catheter occlusion and patient blood stream infections (BSIs). This can be attributed to a lack of knowledge of connector designs and flushing, clamping, and syringe disconnection techniques. This study aimed to assess nurses’ practice, knowledge, attitudes, and key influencers on appropriate care of NCs in an Australian facility and compare these with studies undertaken in the United States in 2011. Methods: A cross-sectional online survey was sent via email with a SurveyMonkey® link to all nurses working in clinical areas (total population sampling approach; approximately 1500 nurses), at an Australian hospital, in 2018. The survey was anonymous and open for 6 weeks. Analysis was with R software. Results: Response rate was approximately 19% (n = 283). Most (89%) of nurses stated that they clean NCs before each access. Only 25% correctly recognised the negative pressure NC, and 79% correctly identified the correct clamping and disconnection sequence. Positive pressure displacement devices were correctly identified by 44% of respondents, with 34% identifying the correct clamping and disconnecting technique. Nurses reported their behaviour was most influenced by local senior nurses. Conclusions: There remains a significant gap in nurses' knowledge of NC device types, as well as the correct clamping and syringe disconnection for both negative and positive displacement NCs. This survey reaffirms that senior nurses are the key influencers of nurses’ adherence to best practice guidelines.