In neonatal intensive care units (NICUs), hygiene and disinfection measures are pivotal to protect neonates from nosocomial infections. This study aimed to evaluate the efficacy of the classical incubators disinfection procedure and to follow-up neonates housed in the incubators for the development of late-onset sepsis (LOS). In a tertiary NICU, 20 incubators were monitored for bacterial contamination at three times: before disinfection, after disinfection, and 24 h after turning on and housing a new neonate. Clinical data of neonates housed in these incubators were retrieved from the medical records. All 20 incubators were contaminated at the 3 times of the study, mainly on mattresses and balances. Coagulase-negative Staphylococci, Enterococcus, and Bacillus-resisted disinfection while enterobacteria and Staphylococcus aureus were eradicated. After 24 h, the bacterial colonisation was similar to the one observed before disinfection. The bacteria isolated on incubators were also found on the caregivers' hands. During the study, two preterm neonates developed a LOS involving a bacterial species that has been previously isolated in their incubator. Pathogenic contaminants persist on incubators despite disinfection and represent a risk for subsequent infection in preterm neonates. Improvements are needed concerning both the disinfection process and incubator design. Procedures of disinfection that are usually recommended in NICUs do not allow for totally eradicating bacteria from incubators. Preterm neonates are housed in incubators colonised with potentially pathogenic bacteria. The control of nosocomial infections in NICUs requires further researches concerning mechanisms of bacterial persistence and ways to fight against environmental colonisation.