Introduction: The skin surface is inhabited by microorganisms responsible for the biochemical balance; some are pathogenic and persist despite hand washing. Objective: To identify, in university students, the bacterial flora resistant to hand washing. Methods: A longitudinal, analytical and observational study was carried out, for which university students were included who previously received educational talks on hand hygiene. Samples were taken before and after hand washing. Results: Of 80 samples analyzed, Staphylococcus epidermidis was identified as the most frequent in 95% of the students and, with hand washing, it was reduced to 60%. The second agent was Staphylococcus saprophyticcus, present in 75% of the cases and, after hand washing, in 35%. E. coli occupied the third position, as it was found in 42.5% of the samples and, after hand hygiene, it was reduced to 17.5%. For transient agents like Klebsiella (P<0.05) and Pseudomonas, handwashing was more effective; however, E. coli was the enterobacterium that remained elevated despite hand hygiene (P=0.01). Conclusion: Some E. coli colonies are resistant to hand washing, similar to resident agents such as Staphylococcus epidermidis, Staphylococcus saprophyticcus and Staphylococcus aureus.