Abstract Nosocomial bloodstream infections (NBSIs), commonly due to central-line associated bloodstream infections (CLABSI), contribute substantially to neonatal morbidity and mortality. We aimed to identify longitudinal changes in incidence of NBSI, microbiological-spectrum, and antibiotic exposure in a large cohort of preterm neonates admitted to the neonatal intensive care unit. We retrospectively assessed differences in annual rates of NBSI (per 1000 patient-days), CLABSI (per 1000 central-line days), and antibiotic consumption (per 1000 patient-days) among preterm neonates (< 32 weeks’ gestation) hospitalized between January 2012 and December 2020. Multi-state Markov models were created to model states of progression of NBSI and infection risk given a central-line on days 0, 3, 7, and 10 of admission. Of 1547 preterm infants, 292 (19%) neonates acquired 310 NBSI episodes, 99 (32%) of which were attributed to a central-line. Over the years, a significant reduction in central-line use was observed (p < 0.001), although median dwell-time increased (p = 0.002). CLABSI incidence varied from 8.83 to 25.3 per 1000 central-line days, with no significant difference between years (p = 0.27). Coagulase-negative staphylococci accounted for 66% of infections. A significant decrease was found in antibiotic consumption (p < 0.001). Probability of NBSI decreased from 16% on day 3 to 6% on day 10. NBSI remains a common problem in preterm neonates. Overall antibiotic consumption decreased over time despite the absence of a significant reduction in infection rates. Further research aimed at reducing NBSI, in particular CLABSI, is warranted, particularly with regard to limiting central-line dwell-time and fine-tuning insertion and maintenance practices.