Background <p>There is a paucity of studies investigating the prevalence of healthcare-associated infections and antimicrobial use at a national level in Kazakhstan. Therefore, this study aims to address this gap by reporting the results of the first nationwide investigation into healthcare-associated infections and antimicrobial use in acute care hospitals.</p> Methods <p>A point-prevalence survey was conducted in 26 acute care hospitals across Kazakhstan in June 2023. The methodology strictly adhered to Protocol 5.3 of the European Centre for Disease Prevention and Control. The sample consisted of 8,076 patients.</p> Results <p>The overall rate of healthcare-associated infections was 2.4%, with 42.9% occurring in surgical wards. Pneumonia was the most common type of healthcare-associated infection (25.0%), followed by surgical site infections (13.2%), and gastrointestinal tract infections (11.8%). Antimicrobial use was reported in 40.2% of patients, with cephalosporins being the most frequently prescribed pharmacological group (59.7%). Resistance to third-generation cephalosporins was the most commonly observed resistance in microorganisms isolated from HAI cases (47.6%).</p> Conclusions <p>Kazakhstan needs to continue implementing infection prevention and control measures and foster the development of antimicrobial stewardship programs tailored to the specific needs of its hospitals.</p>