Introduction. Preterm infants (PTIs) are vulnerable to morbidity, disability, and mortality. They require meticulous care for survival and development in neonatal care units (NCUs). PTI care in NCUs is a collaborative and team effort among different health professionals. However, nurses have a key role for quality care. This study aimed to assess nurses’ PTI care practices across different hospitals in Nepal. Methods. A descriptive cross-sectional study was conducted in NCUs of six randomly selected public hospitals in Nepal. After obtaining ethical approval, structured observation was completed among 40 NCU nurses using a practice checklist. After observation, a self-report questionnaire was administered among 102 nurses. Both descriptive and inferential statistics were used for data analysis. Results. The observation and self-reported mean infant care practices were 73.7% and 70.7%, respectively. The overall practice median score and the interquartile range (IQR) were 4.0 (3.5–4.3) with the highest score of (4.5 [4.1–4.7]) for daily supportive care and the lowest score of (2.8 [2.1–3.5]) for pain management. The care practice was strongly associated with the nurse-infant ratio (adjusted odd ratio (aOR) = 18.52, confidence interval (CI) = 5.83–58.77, and p=<0.001) and training status (aOR = 4.95, CI = 1.59–15.39, and p=0.006). Conclusion. Nurses have adequate practice of thermal care, safe oxygen administration, nutritional care, and infection prevention in NCUs, whereas lacking is found regarding developmental supportive care components (sleep promotion and a supportive sensory environment), parental support, and pain management practices. Consideration of the proper nurse-infant ratio and continued professional development opportunities are essential for practice enhancement in NCUs. These findings might be useful to identify the gaps in PTI care practice in NCUs.