Antisepsis of the hands of medical personnel is one of the most important steps in the process of patient care, since direct contact can cause the cross-transfer of potentially pathogenic microorganisms at surgical sites.This study aimed to analyze the prevalence of microorganisms on the hands of 131 surgeons in a university hospital before the surgical procedure. Swabs were collected from each clinician's hands before and after handwashing. The samples were placed in a transport medium and immediately delivered to a private Clinical Analysis Laboratory from São Luis-Maranhão. The microorganisms were identified by ionization source mass spectrometry and matrix-assisted laser desorption (MALDI-TOF), and antibiotic susceptibility tests (AST) were performed using the Vitek2 automated system. The results showed a high frequency (100%) of microorganisms before handwashing, but after surgical antisepsis, the rate dropped significantly (p<0.05) to 27.5%. Gram-positive cocci and rods were the most common microbes, followed by gram-negative bacilli species. The effectiveness of hand antisepsis was 72.5%. The ideal would be 100% efficacy, that is, 0% microorganisms in all surgeons. It is logical that the presence of any organism, no matter how infrequent it may be, will always represent a great risk of postoperative infection for any patient.