Abstract Background As the only non-European Union (EU) country, Serbia participated in a second point prevalence survey of healthcare-associated infections (HAIs) and antimicrobial use (AMU) organized by the European Centre for Disease Prevention and Control (ECDC) in the EU countries. Here, we aimed to estimate the prevalence of HAI and AMU in patients who had recently undergone a surgery and to compare risk profile, HAI rates, and AMU among surgical patients and non-surgical patients. Methods A national PPS was performed in 65 Serbian acute-care hospitals, in November 2017. In this paper, the data of 61 hospitals for adult acute-care were analyzed. To ensure the comparability of study design we used the Serbian translation of ECDC case definitions and ECDC PPS protocol. The trained infection control staff, led by a hospital coordinator, reviewed medical records to identify HAI active at the time of the survey and AMU. Only inpatients admitted to the ward before 8 a.m. on the day if the survey were included. Results A total of 12,478 patients from 61 hospitals for adult acute-care were eligible for inclusion in this study. Significantly higher proportions of surgical patients were female, belonged to the 60-to-79 age group, and were less severely ill. Also, extrinsic factors (invasive devices, hospitalization at the ICU, and prior antibiotics therapy) were more frequent in surgical patients. Prevalence of HAIs was higher among surgical patients (261/3626; 7.2%) than among non-surgical patients (258/8852; 2.9%) (p