Introduction: Educational interventions can raise awareness and understanding in population groups and may help to achieve changes in behavior. Objective: To determine if the educational handwashing program applied to high-school students reduces the colonization of infectious bacteria on the hands. Methods: A non-randomized quasi-experimental study was carried out using consecutive sampling of the students of the last year of high-school who were divided into an intervention group and a control group. Data were obtained before and after the implementation of the educational program. Samples were taken from the dominant hand for bacterial culture, and a questionnaire was applied that assessed knowledge of hand hygiene before and after the program. The program was aimed at improving the level of understanding and attitudes of students regarding hand hygiene. Results: Of 208 samples obtained, the most frequent agents before and after the educational program were Staphylococcus epidermidis (39% versus 23%) and Staphylococcus aureus (21% versus 15%). The average number of colonies was 236 and 183 respectively (p = 0.35). Conclusion: The educational program managed to improve the level of understanding about hand hygiene; however, it was not enough to reduce bacterial colonization, so other behaviors such as direct supervision should be considered.