The use of instruments designed to assess the nursing practice environment is crucial to improve the quality of nursing care, to anticipate problems and difficulties that may arise in organizations, and allow nurse managers to implement changes and improvements in key areas. In this study, we aimed to evaluate the psychometric properties of the Nursing Work Index—Revised Portuguese version (NWI-R-PT) scale. A quantitative, observational, descriptive, and cross-sectional study was conducted. Methods: The sample consisted of 767 nurses from 4 public Portuguese hospitals. Exploratory and confirmatory factor analysis techniques were used to test the distinct structural models. The scale’s accuracy was evaluated through internal consistency, using Cronbach’s alpha. Results: NWI-R-PT internal consistency was 0.91. The NWI-R-PT model with six factors, namely “Management Support,” “Professional Development,” “Fundamentals of Nursing,” “Nurse–Physician Relationship,” “Endowments,” and “Organization of Nursing Care,” was supported by exploratory and confirmatory factor analysis. The NWI-R-PT scale presents adequate goodness-of-fit indices concerning the final factorial model and the convergent validity. Conclusions: The NWI-R-PT scale has a competent and reliable structure. The scale’s validity is confirmed; therefore, it may be employed in all contexts in clinical practice, research, and nursing management. The NWI-R-PT is a useful and valid instrument to assess the nursing environment in hospitals, primary care, long-term care, and nursing homes. The scale has significance in improving the quality of nursing care and patient safety, the professional development of nurses, and organizational results.