Healthcare-associated sepsis (HAS) is a life-threatening complication in neonatal intensive care. Research into the impact of HAS on mortality adjusted for comorbidities is however limited. We conducted a historical cohort study to evaluate impact of HAS on mortality stratified by birth weight and risk factors for mortality in the HAS cohort. HAS was defined according to the National Institute of Child Health and Human Development criteria. Logistic regression was used to calculate adjusted odds of mortality. Of 5134 admissions, 342 infants developed HAS (6.7 %). Mortality in the total and HAS cohort was 5.6 and 10.5 %, respectively. The majority of HAS was caused by commensals (HAS-COM, 59.4 %) and 40.6 % by recognized pathogens (HAS-REC). Adjusted for comorbidities, "HAS-REC" is only a risk factor for mortality in newborns >1500 g (adjusted odds ratio [aOR] 2.3, confidence interval [CI] 1.1-4.9). Post-hoc analysis identified HAS-REC as an independent risk factor for mortality in infants with gastrointestinal disease (aOR 4.8, CI 2.1-10.8). "Renal insufficiency," "focal intestinal perforation," and "necrotizing enterocolitis" are independent risk factors for mortality in the HAS cohort (aOR 13.5, CI 4.9-36.6; aOR 7.7, CI 1.5-39.2; aOR 2.1, CI 1.0-4.7, respectively). CONCLUSION: For very low birth weight infants (=1500 g), several comorbidities overrule the impact of HAS on mortality. After adjustment for comorbidities, HAS-REC independently predicts in-hospital mortality in heavier infants and in those with gastrointestinal disease. WHAT IS KNOWN: * The relationship between healthcare-associated sepsis and mortality is influenced by the causative pathogen and is confounded by comorbidities. * Research on impact of healthcare-associated sepsis on mortality adjusted for comorbidities is limited as well as research on independent risk factors for mortality in neonates with sepsis. What is New: * We included a large list of comorbidities and stratified risk by birth weight in order to assess the true effect of healthcare-associated sepsis on mortality. * Risk for mortality was calculated for commensal flora and for recognized pathogens as causative micro-organisms.