Introduction: Healthcare-associated infections are concerning adverse events and hand hygiene is considered an essential preventive measure. The objective of the present study was to assess the effect of a correct 3-step hand hygiene technique on reducing of potentially pathogenic microorganisms on hands related to the WHO five moments for hand hygiene. Methodology: A cross-sectional study was performed by means of direct observation involving 60 Intensive Care Units (ICU) and clinical nursing professionals in a Brazilian hospital. Observations were performed in order to ascertain the adherence rate and the correct technique during health assistance. Additionally, microbiological analysis of material collected from the nursing professional’s hands was carried out. Exploratory and inferential analyses were performed on R software and binomial analysis was carried out by using the Z-test. The study was approved by the research ethics committee and covered all the legal principles for the protection of human subjects. Results: Hand hygiene adherence rate was 63.3%. However, only 13.3% of the professionals performed the correct 3-step hand hygiene technique regarding steps and time. Sixty-five microorganisms were isolated, among which 56.9% were coagulase-negative Staphylococcus, 26.2% were Gram-negative bacilli, 7.7% were Enterococcus faecalis, and 6.2% were Candida parapsilosis. There was no presence of potentially pathogenic microorganisms on the nursing professional’s hands who performed the correct three-step technique. Conclusions: Overall correct hand hygiene technique was poor. The results indicated the presence of potentially pathogenic microorganisms at moments in which hand hygiene was mandatory but was not executed or was executed incorrectly. The 3-step hand hygiene technique proved to be effective when correctly performed since there were no microorganism growth. Larger studies are needed to test if these results can be replicated at a larger scale, since streamlining hand hygiene technique yielded encouraging results.