Background: Banknotes are among the objects frequently exchanged between people and can serve as a vehicle for the transmission of infectious agents. Therefore, the present study aimed to isolate and identify potentially pathogenic bacteria on Yemeni banknotes collected from various municipal sources in Aden city, south of Yemen. In addition, the susceptibility of the isolated bacterial species to commonly used antibiotics was tested. Methods: A descriptive cross-sectional study was conducted on 240 Yemeni banknotes of various denominations randomly collected from people from different places in Aden city between November 2019 and February 2020. Swabs from the surfaces of banknotes were streaked onto blood agar, chocolate agar, mannitol salt agar and MacConkey agar and incubated at 37 °C for 48 hours. After isolation, bacterial species were identified and tested for their susceptibility to commonly used antibiotics using established techniques. Results: Out of 240 banknotes examined, 98.3% were found to be contaminated with bacteria, with a 95% confidence interval (CI) ranging from 96 to 99. Among the isolated bacteria, 17.4% were Gram-positive cocci, 33% were Gram-negative bacilli and 49.6% were a combination of both. The mean plate counts of bacterial colonies ranged from 163.6±51.0 colony-forming units (CFU)/ml (95% CI: 147.3–179.9) for banknotes with a denomination of 100 Yemeni rials (YRIs) to 257.1±52.0 CFU/ml (95% CI: 240.5–273.7) for banknotes with a denomination of 50 YRIs. Staphylococcus aureus was the most frequently isolated bacterial species, detected on 66.9% of contaminated banknotes, followed by Escherichia coli (60.6%) and Pseudomonas aeruginosa (42.8%). Regarding antibiotic resistance, most S. aureus isolates showed resistance to ceftazidime (80.1%), followed by ceftriaxone (78.2%), ampicillin (77.6%) and co-trimoxazole (69.3%). On the other hand, most E. coli isolates showed resistance to amoxicillin/clavulanate (84.6%), followed by ceftriaxone (79.7%), ceftazidime (76.9%), ampicillin (58.3%) and ciprofloxacin (58.1%), while most P. aeruginosa isolates showed resistance to ceftriaxone (83.5%), followed by ceftazidime (80.2%) and ciprofloxacin (54.9%). Conclusion: Most banknotes in circulation in Aden city are contaminated with bacteria, predominantly S. aureus and E. coli, with lower denomination banknotes tending to have higher contamination levels. The high contamination rate of banknotes raises concerns about their potential in transmitting infectious diseases and emphasizes the advantages of transitioning to cashless payments. A substantial proportion of bacterial species isolated from banknotes exhibit resistance to commonly used antibiotics, raising concerns about the possible transmission of multidrug-resistant bacteria through the exchange of banknotes. Therefore, it is necessary to raise awareness and improve hygiene practices to minimize the transmission of bacteria and antibiotic resistance through the exchange of banknotes.