Objective: To understand healthcare worker (HCW) perceptions surrounding Staphylococcus aureus transmission and prevention in the neonatal intensive care unit (NICU). Design: Qualitative case study with focus groups. Setting: A level IV, 150-bed NICU at a Midwestern academic medical center that conducts active surveillance and decolonization of S. aureus-positive patients. Participants: NICU HCWs, including bedside nurses, nurse managers, therapy services personnel, pediatric nurse practitioners, clinical fellows, and attending neonatologists. Methods: Semistructured focus group interviews, assembled by occupation, were conducted by 2 study team members. Interviews were video recorded and transcribed. Deductive coding and thematic analyses were performed using NVivo software. Results: In total, 38 HCWs participated in 10 focus groups (1-12 participants each), lasting 40-90 minutes. Four main themes emerged: (1) Methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) are inconsistently described as high risk. (2) Infection prevention interventions are burdensome. (3) Multiple sources of transmission are recognized. (4) opportunities exist to advance infection prevention. HCWs perceived MSSA to be less clinically relevant than MRSA. Participants expressed a desire to see published data supporting infection prevention interventions, including contact precautions, environmental cleaning, and patient decolonization. These practices were identified to be considerable burdens. HCWs perceived families to be the main source of S. aureus in the NICU, and they suggested opportunities for families to play a larger role in infection prevention. Conclusions: These data highlight opportunities for HCW and parental education, research, and reevaluating interventions aimed at improving infection prevention efforts to reduce the burden of S. aureus in NICU settings.