Introduction: Healthcare-associated infections (HCAIs) are an important cause of morbidity and mortality in hospitalized patients. In this study, we aimed to investigate the effect of prophylactic enteral probiotic supplementation on necrotizing enterocolitis (NEC), mortality, and HCAIs in infants hospitalized in the neonatal intensive care unit (NICU). Materials and Methods: In the first six months of the one-year study, the >1000-gram infants hospitalized in the NICU constituted the probiotic-free group (group 1, n= 119). In the second six months of the study, the >1000-gram infants admitted to the unit were given daily routine prophylactic enteral probiotic (Bifidobacterium animalis (BB-12) 1 x 109 CFU and Streptococcus thermophilus (TH-4) 1 x 108 CFU, Bifiform®) supplementation (group 2, n= 78). HCAIs and mortality rates were compared between both groups. Results: One hundred ninety-seven patients were included in the study and their demographic characteristics were analyzed. The incidence of NEC was significantly lower in group 2 (0/78 patients vs. 6/119 patients; p= 0.044). As a secondary outcome, the number of HCAIs (p= 0.039) and the proportion of HCAIs with the causative agent in culture (p= 0.018) were lower in group 2 compared to the group not receiving probiotics. The most common HCAI subgroups in NICU were bloodstream infection, central line-associated bloodstream infection, and NEC. The HCAIs incidence density was 22.05 in group 1 and 14.02 in group 2, which was not statistically significant. However, when HCAIs incidence densities were analyzed according to subgroups, a statistically significant difference was observed in the rate of central catheter-related bloodstream infections (38.78 vs. 5.69, p≤ 0.001). The rate of HCAIs caused by gramnegative microorganisms was lower in group 2 (15/119 vs. 3/78; p= 0.029). Conclusion: We observed a statistically significant reduction in the incidence of NEC and overall mortality in premature infants given prophylactic probiotics. We also found a significant reduction in HCAIs and gram-negative sepsis in the same group.