The aim of this study was to objectively assess the hand hygiene performance of medical students. Hand rubbing technique was evaluated by employing innovative UV-light-based imaging technology, identifying patterns and trends in missed areas after applying WHO’s six-step protocol. This specially designed hand hygiene education and assessment program targeted 1,344 medical students at two distant sites in Central Europe. Students were introduced to a short video, presenting the basics of hand hygiene, and then received further demonstration from professional trainers, focusing on the correct execution of WHO’s six-step technique. To verify the acquired skill, participants rubbed their hands with UV-marked alcohol-based solution. Digital images of the hands were recorded under UV light, followed by computer evaluation and assessment. Immediate objective visual feedback was given to the participants showing missed areas on their hands. The statistical analysis of missed spots was based on retrospective expert-driven manual evaluation. Significant difference in rubbing quality was found between female and male participants [35.3% (CI 95%: 33–38%) versus 29.0% (CI 95%: 27–31%), p < 0.001], dominant and non-dominant hands [43.4% (CI 95%: 39–48%) versus 34.9% (CI 95%: 32–38%), p = 0.002], and various zones of the hands’ dorsal side. Based on the participants’ feedback and the evaluation of the infection control specialists, it can be stated that the identification of typically missed patterns and the instant visual feedback have a vital role in improving the hand hygiene technique of prospective medical staff.