OBJECTIVE: To assess the knowledge, beliefs, and practices of neonatal intensive care unit (NICU) healthcare workers (HCWs). DESIGN: Self-administered survey. SETTING: A 55-bed NICU. PARTICIPANTS: NICU HCWs (N = 215). RESULTS: The response rate was 68%. Ninety-two percent knew central venous catheters (CVCs) should be capped, clamped, or connected to running fluids at all times. Ninety-five percent knew when to change gloves. Thirty-one percent knew the recommended duration for handwashing. Most HCWs believed sterile technique in CVC care (96%), gloves (91%), and handwashing (99%) prevent nosocomial infection (NI). Sixty-seven percent used sterile barriers to insert CVCs, 76% reported wearing gloves, 81% reported routine handwashing, 35% knew that bacterial hand counts are higher with rings, 30% knew that long fingernails are associated with higher gram-negative bacterial hand contamination, and 35% knew that artificial fingernails are associated with higher gram-negative bacterial hand contamination. Most (93%) believed HCWs can affect outcomes of patients with NIs. Fewer believed rings (40%), artificial fingernails (61%), and long fingernails (48%) play a role in NIs, or that policies concerning number of rings (50%), cutting fingernails (35%), or prohibiting artificial fingernails (47%) would prevent NIs. Sixty-one percent of HCWs regularly wore at least one ring to work, 56% wore their fingernails shorter than the fingertip, and 8% wore artificial fingernails. CONCLUSIONS: A disconnect existed between CVC knowledge and beliefs and practice. HCWs did not know the relationship between bacterial hand counts and rings and fingernails, and did not believe rings or long or artificial fingernails increased the risk of NIs.