<p>Bats are a diverse order of mammals that play an important role in ecosystem services. Numerous recent studies have focused on bats’ role in the emergence and transmission of zoonotic pathogens, particularly viruses, but relatively fewer studies have investigated antimicrobial resistance in bacteria harbored by bats. Here, we estimate the prevalence and antibiotic profiling of <i>Staphylococcus (S.) aureus</i> in oral and rectal samples from bats in Northern Pakistan and assess factors associated with carriage of these bacteria by bats. Two hundred individual bats of five species: <i>Pipistrellus javanicus</i> (n = 17), <i>Pipistrellus pipistrellus</i> (n = 10), <i>Rhinopoma microphyllum</i> (n = 48), <i>Rousettus leschenaultii</i> (n = 124), and <i>Scotophilus kuhlii</i> (n = 1) were captured for non-lethal collection of samples to isolate <i>S. aureus</i>. Bats sampled were collected from two different provinces of Pakistan, specifically&#xa0;Khyber Pakhtunkhwa and Punjab, in August 2018 and from Sheikhupura Fort in October 2019. Bats were sampled from three sites: a natural cave, a man-made castle, and an animal shed, in Khyber Pakhtunkhwa and Punjab provinces. Out of 400 samples, 58 swabs were positive for <i>S. aureus,</i> including 34 rectal and 24 oral swabs. Prevalence of <i>S. aureus</i> carriage varied significantly among species (0–100%), with <i>Rousettus leschenaultii</i> exhibiting the highest prevalence. In addition, prevalence varied significantly among habitats but not between sex, age class, or reproductive status. <i>S. aureus</i> isolates showed resistance to tetracycline, gentamicin, and erythromycin and carried resistant genes such as <i>Tet</i>K, <i>Tet</i>M, <i>erm</i>A, and <i>aacA</i>-D. In this context, it is imperative to prioritize initiatives aimed at educating local communities about reducing exposure to antibiotic-resistant bacteria disseminated by bats and concurrently fostering awareness regarding the critical role bats play in our ecosystem.</p>