Background: Healthcare workers are at the forefront of the recent COVID-19 pandemic. In this study, we aimed to investigate the changes in handwashing behavior of healthcare workers before and during the pandemic. Methods: A survey was conducted at an obstetrics and gynecology clinic situated in Trabzon, Turkey. The survey contained 30 questions divided into two sections. The first 5 questions of which comprised the participants’ demographic data such as their age, body mass index, gravidity, personal history, and occupation. The first and second sections of the questionnaire form covered the questions concerning habits of participants pre-pandemic and during the pandemic, respectively. The total handwashing scores before and during the pandemic were calculated by summing the answers for 25 questions relating to handwashing habits. Independent t-tests were performed for normally distributed data. Results: The data of 98 participants were analyzed. While there was no difference in handwashing scores between doctors (83.44 \pm 6.44%) and midwives (86.26 \pm 13.73%), there was a statistically significant difference between doctors and nurses (90.04 \pm 11.08%) (p = 0.02). The results also showed that there was a significant difference (p = 0.03) in daily handwashing frequency of doctors and nurses before and during the pandemic. Conclusions: Healthcare professionals attached importance to hand hygiene before the pandemic, and this is being emphasized even more during the pandemic with a significant difference. The highest increase was accounted for nurses.