Objective: To assess the nurse professional practice environment of neonatal units and its relationship with the levels and main sources of occupational stress. Method: Cross-sectional descriptive, exploratory, correlational study performed with nursing professionals of neonatal units of four public hospitals. A sociodemographic/professional questionnaire, the Brazilian version of Practice Environment Scale, and the Work Stress Scale were applied. In the analysis, mean, standard deviation, and Pearson chi-squared, Likelihood Ratio, and Mann Whitney U tests were adopted for association among variables. Results: Participating professionals amounted to 269. The practice environment was evaluated as favorable by more than half of the sample (63.6%), showing a significant statistical association which was inversely proportional with occupational stress (p < 0.001). The insufficient number of professionals for quality care was the major source of stress for nursing technicians, whereas teamwork with doctors was the predominant factor for the evaluation of environment quality and high stress levels of nurses. Conclusion: Unfavorable practice environments increase the stress levels of nursing professionals in neonatal units and may compromise patient safety.