Abstract Background Healthcare-associated infections (HAIs) in children contribute to elevated morbidity and mortality rates. While the surveillance of antibiotic-resistant bacteria involved in HAIs is important, there is limited data available in Latin America. The present study aimed to evaluate the antibiotic resistance levels in bacteria isolated from hospitalized patients with HAIs in a pediatric referral center in Lima – Peru. Methods Surveillance data of HAIs from November 2021 to June 2023 among pediatric patients at Instituto Nacional de Salud del Niño San Borja was prospectively collected and reviewed. The dataset included comprehensive information on the clinical and epidemiological characteristics of patients, bacteria isolation sites and identification, as well as resistance profiles to 13 antibiotics. HAIs included were central line-associated bloodstream infections (CLABSI), catheter-associated urinary tract infections (CAUTI) and ventilator-associated pneumonia (VAP). Results We included 143 bacterial isolates from pediatric patients hospitalized with HAIs, of which 53.85% were male (n=77). The median age of the patients was 7 months (IQR: 2 -36). A total of 44.76% (n=64) had congenital diseases and 20.28% (n=29) had cancer. Most of the isolates were collected from pediatric Intensive Care Unit (n=104, 72.73%). The median time from the hospital admission to the appearance of HAIs was 17 days (IQR: 7-28). The most frequent isolates were Gram negative bacteria: Klebsiella pneumoniae (n=33), Pseudomonas aeruginosa (n=34) and Escherichia coli (n=24). Among Gram positive bacteria, Staphylococcus epidermidis (n=5) and Staphylococcus aureus (n=5) were the most frequent species. Regarding the site of isolation, the primary etiological agent in CLABSI was K. pneumoniae (n=18); in CAUTI, E. coli (n=13) and in VAP P. aeruginosa (n=9). Multidrug-resistant (MDR) isolates were identified, mainly among K. pneumoniae (62.50%), E. coli (54.55%), and P. aeruginosa (26.74%). Difficult-to-treat resistance was only present in 2.94% of P. aeruginosa isolates. Overall resistance to piperacillin-tazobactam was below 16%. Resistance to cephalosporins was higher than 40% for K. pneumoniae and E. coli isolates, with frequencies exceeding 60% for cefuroxime, ceftazidime, ceftriaxone, cefepime and cefoperazone/sulbactam. E. coli and K. pneumoniae resistance to carbapenems was below than 10%; however, P. aeruginosa resistant isolates were identified in 38.24% and 39.39% for meropenem and imipenem, respectively. Ciprofloxacin resistance was frequent among E. coli (70%) and K. pneumoniae (60.87%), whereas only 26.67% of P. aeruginosa. Amikacin resistance was detected in K. pneumoniae and P. aeruginosa only, with a frequency of 23.08% and 20%, respectively. Conclusion Alarming levels of antibiotic-resistant bacteria were found in HAIs among pediatric patients. Our results highlight the need of supporting initiatives for HAIs control, developing institutional guidelines for prevention and strengthening antibiotic stewardship in pediatric patients. Table 1: Antibiotic resistance profiles of the main microorganisms of healthcare-associated infections (November 2021 – June 2023)