Background: Surveillance is essential to all aspects of management of healthcare-associated infections (HAIs) in critically ill children, where data are limited. We conducted an active surveillance study to elucidate epidemiology, resistance, antimicrobial treatment practices and outcomes of pediatric intensive care unit-acquired HAIs in a southern European country., Methods: Four Greek pediatric intensive care unit encounters (153 patients, 2183 patient-days) during a 6-month period participated using the European Centre for Disease Prevention and Control HAI-net ICU (v2.2) protocol. Bloodstream infections and device-associated HAIs were recorded. Clinical severity, isolated pathogens, antimicrobial resistance and antibiotic prescriptions were collected on a daily basis. Mortality and excess length of stay due to HAI were also assessed., Results: Overall rate of HAIs was 18.3 per 1000 patient-days. Aggregate rates for device-associated HAI were: catheter-related bloodstream infection 2.32, intubation-associated pneumonia 10.5, and catheter-associated urinary tract infection 4.6 per 1000 device-days. Children with HAI (n = 28, 18.3%) had higher severity of illness (Pediatric Risk Mortality Score 7.5 vs. 4, P < 0.001), longer hospitalization (23 vs. 6 days, P < 0.001), but not higher mortality, compared with those without. Most frequent recovered pathogens were Klebsiella pneumoniae (40%), Pseudomonas aeruginosa (22.5%), Acinetobacter baumannii (12.5%), with respective carbapenem resistance 50%, 44% and 80%, and Staphylococcus aureus (12.5%). Total antibiotic use was 2142 days of treatment per 1000 patient-days., Conclusions: Our study, based on the updated ECDC HAI-net ICU (v2.2) protocol, effectively addresses the significant burden of HAIs in critically ill children in Greece. Using a well-standardized system facilitates inter- and intra-countries reliable recordings and comparative assessments of infection control programs., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)