Objective:Investigation of the origin of aSerratia marcescensoutbreak in a neonatal intensive care unit.Design:Retrospective case–control study.Setting:Regional level 3 perinatal center in Germany.Patients:This study included 4S. marcescens–positive and 19S. marcescens–negative neonates treated between February 1 and February 26, 2019, in the neonatal intensive care unit.Methods:A case–control study was performed to identify the source of the outbreak. The molecular investigation of S. marcescens isolates collected during the outbreak was performed using pulsed-field gel electrophoresis and next-generation sequencing.Results:The retrospective case–control study showed a significant correlation (P < .0001) between S. marcensens infection or colonization and consumption of donor milk that had tested negative for pathogenic bacteria from a single breast milk donor. Pulsed-field gel electrophoresis and next-generation sequencing retrospectively confirmed an S. marcescens strain isolated from the breast milk of this donor as the possible origin of the initial outbreak. The outbreak was controlled by the implementation of an infection control bundle including a multidisciplinary infection control team, temporary nutrition of infants with formula only and/or their mother’s own milk, repeated screening of all inpatients, strict coat and glove care, process observation, retraining of hand hygiene and continuous monitoring of environmental cleaning procedures.Conclusions:Low-level contaminated raw donor milk can be a source of infection and colonization of preterm infants with S. marcescens even if it tests negative for bacteria.