BackgroundSurveillance for healthcare-associated infections (HAI) is a priority in the neonatal intensive care unit (NICU), given the critical immune status of patients. The aim of this study was to assess surface bacterial contamination before and after improving cleaning and disinfection practices.Materials and methodsThis was a cross-sectional study conducted in March 2018. Surface samples were taken from the same areas in three steps: after cleaning, after "improved" cleaning, and after terminal disinfection using hydrogen peroxide vapor (VHP). Sampling and culture was carried out according to standard ISO14698-1: 2004. Results interpretation was based on the thresholds defined by good hospital pharmacy practice. Statistical analysis was performed by SPSS 21.0 and a P-value < 0.05 was considered to be significant.ResultsIn total, 290 samples were taken from different zones: fixed equipment (69%), aseptic washbasins (12%), pneumatic system (9%), computer equipment (6%) and mobile equipment (4%). Prevalence of non-compliances after cleaning and disinfection was 75%, 10% after “improved” cleaning, and 0% after automated VHP (P < 0.0001). Median of CFU was 24[EI (0–625)] after standard cleaning, 2[EI (0–35)] after “improved” cleaning and 0 [EI (0–3)] after VHP (P < 0.0001). Isolated germs werecoagulase-negative Staphylococcus (31.2%), Acinetobacter baumannii (26%), Staphylococcus aureus (19.5%), Pseudomonas aeruginosa (9%), Klebsiella pneumoniae (9%), E. coli (4%) and Enterobacter sp (1.3%).ConclusionImproved cleaning and disinfection practices associated to VHP give microbiological satisfactory results. It is important to educate cleaning staff for effective surface cleaning and disinfection operations to control HAI.