Introduction: Compliance with Hand hygiene (HH) has been considered as a simple and the most efficient strategy to reduce hospital-acquired infections (HAIs). Compliance with this practice is not often assessed in Nigeria and thus posed a significant risk to the quality of care and patient safety and especially in this era of the COVID-19 pandemic. The study aimed to assess neonatal nurses’ compliance to “my five moments for HH”. Method: A cross-sectional observational study was carried out in a Nigerian federal hospital, using an adapted version of a standardized observational WHO tool of ‘’my five moments of HH’’. A Chi-square test was employed to examine the relationship of HH compliance across units, shifts, and “my five moments for HH” opportunities. Results: The observer recorded 425 HH opportunities and the total HH compliance rate was (62.8%). There was no association between the units, working shifts, and nurses’ HH compliance. Compliance to HH was dependent on the five moments of HH (p < 0.000), better compliance was seen after body fluid exposure (100%), followed by after newborn contact (86.3%), and after contact with newborn surroundings (63.4%), while compliance before newborn contact (28.5%), and before an aseptic procedure (9.1%) were poor. Conclusion: The findings of this study showed a significant gap with the HH compliance among neonatal nurses, suggesting multiple opportunities for neonatal infections despite the current awareness of HH in the Covid-19 pandemic. Hence, there is a need to intensify infection control practices in Nigeria, especially on HH practice.