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. A laboratory-based study was conducted with a pragmatic approach. Samples were handled with different types of gloves and bare hands, and cytotoxicity assays were performed using the agar diffusion method. Additionally, microbiological analysis was carried out on samples handled without gloves. The results showed no cytotoxic effects in any of the samples, regardless of the type of glove used. All samples of gloves exhibited toxicity when placed directly onto the cell layer, but the toxicity was graded as moderate (Grade 2) and did not represent a toxicological risk to the cells. The microbiological analysis showed microbial growth in all samples, but no microorganisms were recovered after autoclaving. The sterilization control group did not show any microbial growth. The study found that there were no differences in cytotoxic responses between the use of different types of gloves and bare hands. However, the use of gloves increases the cost of the process and waste generation, and poses a potential allergenic risk to latex. The results suggest that the preparation of surgical instruments without gloves is equally safe and may be a better option to reduce the risk of latex allergy and environmental impact. The study also highlights the importance of hand hygiene in the preparation process, as the microbial load on the hands of staff can contribute to the contamination of surgical instruments. The findings indicate that the use of gloves is not necessary for the preparation of clean surgical instruments and that the current recommendation in Brazil for the use of gloves may not be justified. The study concludes that the preparation of surgical instruments without gloves is the ideal recommendation.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. A laboratory-based study was conducted with a pragmatic approach. Samples were handled with different types of gloves and bare hands, and cytotoxicity assays were performed using the agar diffusion method. Additionally, microbiological analysis was carried out on samples handled without gloves. The results showed no cytotoxic effects in any of the samples, regardless of the type of glove used. All samples of gloves exhibited toxicity when placed directly onto the cell layer, but the toxicity was graded as moderate (Grade 2) and did not represent a toxicological risk to the cells. The microbiological analysis showed microbial growth in all samples, but no microorganisms were recovered after autoclaving. The sterilization control group did not show any microbial growth. The study found that there were no differences in cytotoxic responses between the use of different types of gloves and bare hands. However, the use of gloves increases the cost of the process and waste generation, and poses a potential allergenic risk to latex. The results suggest that the preparation of surgical instruments without gloves is equally safe and may be a better option to reduce the risk of latex allergy and environmental impact. The study also highlights the importance of hand hygiene in the preparation process, as the microbial load on the hands of staff can contribute to the contamination of surgical instruments. The findings indicate that the use of gloves is not necessary for the preparation of clean surgical instruments and that the current recommendation in Brazil for the use of gloves may not be justified. The study concludes that the preparation of surgical instruments without gloves is the ideal recommendation.