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 and Phoenix-BD 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%. The gram-positive species most detected were Staphylococcus spp. and Micrococcus luteus, representing 83.9%, followed by gram-negative species, Stenotrophomonas maltophilia, Acinetobacter baumanii, Pseudomonas aeruginosa, Pseudomonas gessardi, Pantoea septica, Serratia marcescens, and Burkholderia lata. The effectiveness of hand antisepsis was 72.5%, demonstrating that surgeons’ hands are an important source of microorganisms that can cause infections in hospitalized patients in different care settings.