We aimed to evaluate the isolation of strains contained in the Infloran™ probiotic preparation in blood cultures and its efficacy in reducing necrotizing enterocolitis (NEC) and late-onset sepsis (LOS) in extremely preterm infants. Routine use of probiotics was implemented in 2008. Infants born at <28 weeks gestational age were prospectively followed and compared with historical controls (HC) born between 2005 and 2008. Data on sepsis due to any of the two probiotic strains contained in Infloran and rates of LOS and NEC were analysed. A total of 516 infants were included. During the probiotic period (PC), none of the strains included in the administered probiotic product were isolated from blood cultures. Probiotic administration was associated with an increase in NEC stage II or higher (HC 10/170 [5.9%]; PC 46/346 [13.3%]; P=0.010). Surgical NEC was 12.1% in PC (42/346) versus 5.9% (10/170) in HC (P=0.029). Adjusting for confounders (sex, gestational age, antenatal steroids and human milk) did not change those trends (P=0.019). Overall, clinical LOS and the incidence of staphylococcal sepsis were lower in PC (172/342, 50.3, and 37%, respectively) compared with HC (102/169, 60.3 and 50.9%, respectively) (P=0.038 and P=0.003, respectively). No episodes of sepsis attributable to the probiotic product were recorded. The period of probiotic administration was associated with an increased incidence of NEC after adjusting for neonatal factors, but also with a reduction in the LOS rate.