Background: Healthcare worker (HCW) uniforms and cell phones are involved in pathogen transmission. This study aimed to characterize pathogenic microorganism isolates from HCW uniforms and cell phones. Methods: Gram-negative microorganisms were recovered from HCW uniforms and cell phones. Antimicrobial susceptibility and the presence of extended-spectrum β-lactamases (ESBL) and carbapenemases were determined. Results: Escherichia coli was the most prevalent microorganism. Overall, high levels of resistance to cephalosporins, quinolones, co-trimoxazole and colistin were found. ESBL were mainly related to blaCTX-M-15 and blaSHV- genes. Carbapenem-resistant isolates presented as blaKPC or blaNDM. Conclusions: High levels of antimicrobial resistance, including colistin, were detected. Therefore, strategies are urgently needed to prevent bacterial dissemination.