This study aimed to determine if there are differences in the safety of preparing clean surgical instruments using different types of gloves and bare hands, and to evaluate the microbiological load of these preparations without gloves. The research was conducted in a laboratory setting with a pragmatic approach. Samples were handled with different types of gloves (powdered latex, non-powdered latex, vinyl, and nitrile) and bare hands. Cytotoxicity assays were performed using the agar diffusion method, and microbiological analysis was conducted after handling without gloves. The results showed no cytotoxic effects from any of the samples. All microbiological cultures grew microorganisms, but no microorganisms were recovered after autoclaving. The study concluded that there were no significant differences in cytotoxic responses between using different types of gloves and bare hands in handling clean surgical instruments, which could pose iatrogenic risks. The use of gloves increases costs and generates waste, and there is a potential risk of latex allergies. The study recommends that the preparation of surgical instruments should be done without gloves to minimize these risks and environmental impacts.This study aimed to determine if there are differences in the safety of preparing clean surgical instruments using different types of gloves and bare hands, and to evaluate the microbiological load of these preparations without gloves. The research was conducted in a laboratory setting with a pragmatic approach. Samples were handled with different types of gloves (powdered latex, non-powdered latex, vinyl, and nitrile) and bare hands. Cytotoxicity assays were performed using the agar diffusion method, and microbiological analysis was conducted after handling without gloves. The results showed no cytotoxic effects from any of the samples. All microbiological cultures grew microorganisms, but no microorganisms were recovered after autoclaving. The study concluded that there were no significant differences in cytotoxic responses between using different types of gloves and bare hands in handling clean surgical instruments, which could pose iatrogenic risks. The use of gloves increases costs and generates waste, and there is a potential risk of latex allergies. The study recommends that the preparation of surgical instruments should be done without gloves to minimize these risks and environmental impacts.