This study aims to assess the association of nursing overtime, nurse staffing, and unit occupancy with health care-associated infections (HCAIs) in the neonatal intensive care unit (NICU). A 2-year retrospective cohort study was conducted for 2,236 infants admitted in a Canadian tertiary care, 51-bed NICU. Daily administrative data were obtained from the database "Logibec" and combined to the patient outcomes database. Median values for the nursing overtime hours/total hours worked ratio, the available to recommended nurse staffing ratio, and the unit occupancy rate over 3-day periods before HCAI were compared with days that did not precede infections. Adjusted odds ratios (aOR) that control for the latter factors and unit risk factors were also computed. A total of 122 (5%) infants developed a HCAI. The odds of having HCAI were higher on days that were preceded by a high nursing overtime ratio (aOR, 1.70; 95% confidence interval [95% CI], 1.05-2.75, quartile [Q]4 vs. Q1). High unit occupancy rates were not associated with increased odds of infection (aOR, 0.85; 95% CI, 0.47-1.51, Q4 vs. Q1) nor were higher available/recommended nurse ratios (aOR, 1.16; 95% CI, 0.67-1.99, Q4 vs. Q1). Nursing overtime is associated with higher odds of HCAI in the NICU.