Background: Hand hygiene is essential in healthcare to prevent infections and ensure patient safety. The World Health Organization highlights hand hygiene as a key measure to reduce healthcare-associated infections. However, adherence to hand hygiene protocols is often inadequate, with a low compliance rate. First-year nursing students are at a critical stage in developing foundational practices. Traditional teaching methods have shown limited success in achieving long-term adherence, necessitating the need for more interactive and engaging teaching methods. Therefore, this study aims to evaluate the effects of an interactive lecture on the handwashing knowledge of first-year nursing students. Methods: This study employed a quasi-experimental one-group pretest-posttest design involving 60 first-year Thai nursing students at Boromarajjonani College of Nursing. After four weeks of interactive lectures, participants completed a pretest and posttest to assess changes in hand hygiene knowledge and understanding of WHO Five Moments for Hand Hygiene. The intervention included visual demonstrations, hands-on practice, and real-time feedback. Data were analyzed using descriptive statistics and paired t-tests to determine the intervention’s effectiveness. Results: The study showed significant improvements in both handwashing knowledge and understanding of WHO Five Moments for Hand Hygiene post-intervention. The mean score for handwashing knowledge increased from 5.41 (SD = 1.06) to 6.62 (SD = 1.10) (t(59) = 7.154, p < 0.001). Similarly, the mean score for understanding WHO Five Moments for Hand Hygiene improved from 4.32 (SD = 0.68) to 4.97 (SD = 0.84) (t(59) = 5.60, p < 0.001). Correct responses to specific handwashing questions also increased significantly. Conclusions: The interactive handwashing lecture intervention significantly improved nursing students’ knowledge and understanding of hand hygiene practices. These findings support the integration of interactive educational strategies in nursing curricula to improve hand hygiene compliance and patient safety. Future research should address study limitations, such as sample size and long-term knowledge retention, to further validate these results.