BackgroundAbout 1.4 million people worldwide are affected by hand hygiene-associated infections from healthcare providers, and altered perceptions might be the contributory factor. Objective To assess nurses' perceptions and adherence to the "five moments of hand hygiene" in selected units at a University Teaching Hospital in Rwanda. Methods A descriptive cross-sectional design was used. A sample size of 84 nurses was selected using convenience sampling. The perceptions survey questionnaire and the observation checklist was used to collect data. Descriptive and inferential statistics were used to analyse the data. ResultsSixty-nine (82.1%) had a positive perception of susceptibility and severity, and 75 (89.2%) had a positive perception of the benefits of hand hygiene. All 84 (100%) and 67 (79.8%) had a negative perception of perceived barriers and action (cues) to hand hygiene; respectively. Highest adherence rate (82.1%) was after body fluid exposure risk, lowest before touching a patient (27.4%), with an average adherence rate of 53.6% to the "five moments." Qualification was associated with nurses' perceptions (p =.002) and department of work with adherence to hand hygiene (p =.001). ConclusionThe overall perceptions of nurses were negative with inadequate adherence to hand hygiene practices. Therefore, this necessitates new strategies and reinforcement of hand hygiene among the nurses.Rwanda J Med Health Sci 2019;2(2):160-171. _______________________________________________________________________