Background: Needleless connectors (NCs) are essential devices designed to provide safe, needle-free connection between venous access devices, syringes and infusions. There is a variety of designs, and associated decontamination products and practices; the resulting confusion can cause detrimental patient outcomes. This study aimed to explore nurses’ attitudes, techniques, and practices around the use and decontamination of NCs in clinical practice. Methods: Qualitative inquiry was conducted with seven focus groups of 4–6 participants each in the cancer and surgical units of a large tertiary hospital in Australia between January and March 2019. Participants comprised nurses who had taken part in a recent clinical trial of NC decontamination. Focus group sessions were recorded, transcribed and synthesised using content analysis. Results: Seven focus groups were conducted (total, N = 30 participants), lasting 16–20 min. Six major themes were identified surrounding needleless connector use and decontamination: ‘safety and utility’; ‘terminology and technological understanding’; ‘clinical practice determinants’; ‘decontamination procedures and influencers’; ‘education and culture’; and ‘research and innovation’. Conclusion: The participants articulated positive attitudes towards needleless connector use for needle-stick and infection prevention, however rationales for care and maintenance practices demonstrated limited understanding of guidelines (e.g., disinfection time) and specific NC function (e.g., positive, negative pressure). The findings indicated the need for targeted, standardised needleless connector education, to enhance staff confidence, improve consistency of care and ensure patient safety.