Objective To evaluate the necessity of routine fluconazole prophylaxis in very preterm infants/very low birth weight (VLBW) infants in neonatal intensive care units (NICUs) with a very low incidence of late-onset invasive fungal infection (IFI), and to inform clinical practice. Methods A retrospective cohort study using propensity score matching was conducted including eligible very preterm infants/VLBW infants admitted to the NICUs of West China Second Hospital, Sichuan University and Sichuan Provincial Children's Hospital from January 2018 to December 2023. Infants were categorized into fluconazole and non-fluconazole groups according whether they received fluconazole prophylaxis. The primary outcome was the incidence of late-onset IFI. Results A total of 1 961 infants were enrolled, with an overall late-onset IFI incidence of 0.25% (5/1 961). After propensity score matching, 442 infants were included in each group. No statistically significant differences were observed between the fluconazole and non-fluconazole groups in the incidence of late-onset IFI (0.45% vs 0.23%), mortality (2.04% vs 0.90%), retinopathy of prematurity requiring surgery (6.11% vs 4.98%), intraventricular hemorrhage grade ≥2 (9.28% vs 6.56%), necrotizing enterocolitis stage ≥2 (10.18% vs 8.14%), or bronchopulmonary dysplasia (36.20% vs 36.88%), nor in length of hospital stay (48.5 days vs 50.5 days) (all P>0.05). Conclusions In NICUs with a very low incidence of late-onset IFI, routine fluconazole prophylaxis is not recommended for very preterm infants/VLBW infants.