To describe barriers and facilitators to the adoption of recommended infection prevention and control (IPC) practices among healthcare workers (HCWs).A qualitative research design was used. Individual semistructured interviews with HCWs and observations of clinical practices were conducted from February to May 2018 in 8 care units of 2 large tertiary-care hospitals in Montreal (Québec, Canada).We interviewed 13 managers, 4 nurses, 2 physicians, 3 housekeepers, and 2 medical laboratory technologists. We conducted 7 observations by following IPC nurses (n = 3), nurses (n = 2), or patient attendants (n = 2) in their work routines. Barriers to IPC adoption were related to the context of care, workplace environment issues, and communication issues. The main facilitator of the IPC adoption by HCWs was the "development of an IPC culture or safety culture." The "IPC culture" relied upon leadership support by managers committed to IPC, shared belief in the importance of IPC measures to limit healthcare-associated infections (HAIs), collaboration and good communication among staff, as well as proactivity and ownership of IPC measures (ie, development of local solutions to reduce HAIs and "working together" toward common goals).Adoption of recommended IPC measures by HCWs is strongly influenced by the "IPC culture." The IPC culture was not uniform within hospital and differences in IPC culture were identified between care units.