Background. Health care-associated infections (HAIs) significantly contribute to morbidity and mortality, particularly in intensive care units (ICUs). Among them, medical device-associated infections (MDAIs) such as ventilatorassociated pneumonia (VAP), catheter-associated urinary tract infection (CAUTI), and central line-associated bloodstream infection (CLABSI) pose serious infection control challenges. Considering this burden, the current study was aimed to analyze the epidemiology, microbial profile, antibiotic resistance patterns and genotyping of MDAIs in a tertiary care ICU. Materials and methods. A cross-sectional study was conducted from March 2020 to September 2021 in the ICUs of a tertiary care hospital. A total of 1,025 clinical samples (endotracheal aspirates, central venous catheter blood, and Foley’s catheter urine samples) were collected from patients with suspected MDAIs. Conventional microbiological methods were used for identification and characterization of isolates. Carbapenem-resistant isolates posing HAIs were analyzed for blaIMP and blaOXA-48 genes using polymerase chain reaction (PCR). Results. Of the 1,025 samples, 497 (48%) were culture-positive. Among MDAIs, VAP was most common (66.5%), followed by CAUTI (33%) and CLABSI (0.002%). Klebsiella pneumoniae (23%) was the predominant isolate, followed by non-fermenting Gram-negative bacilli (17%), Escherichia coli (14%), and Pseudomonas aeruginosa (14%). Carbapenem resistance were identified among 12 isolates; 41.6% carried blaIMP, and 16.6% carried blaOXA-48 genes. Conclusion. VAP remains the leading MDAI, with Klebsiella pneumoniae as the major pathogen. Gene-level detection of carbapenem resistance highlights the growing antimicrobial resistance in ICUs. Strengthening antimicrobial stewardship and adherence to infection control bundles are vital to minimizing MDAI rates. © 2025, Amaltea Medical Publishing House. All rights reserved.