Vaccination plays a crucial role in controlling the rate of coronavirus transmission and infectivity. Healthcare professionals are, in fact, at the greatest risk of contracting coronavirus due to their proximity and prolonged exposure to infected patients; this certitude alone enhances the stress and anxiety among patients and professionals alike. In this study, we aimed to assess the levels of anxiety experienced by healthcare professionals in their practices before and after getting vaccinated. This cross-sectional study was carried out in 2021. An electronic survey was distributed among the non-vaccinated and vaccinated healthcare workers. The survey consisted of the following parts: demographic characteristics, coronavirus-related questions, questions related to the specific field of healthcare professions, general anxiety questions, and working-hour-related questions. The Modified General Anxiety Scale (GAD-7) was used along with the paired t-test, Mann–Whitney U test, and Spearmen’s test for comparison. p ≤ 0.05 was considered statistically significant. A total of 798 healthcare professionals participated in the study. In this study, the majority of participants were females, with 598 (74.9%) being between the ages of 21 and 30, and 646 (80.9%) participants were graduates, with the majority being dentists. Non-vaccinated healthcare professionals had severe anxiety (30.9%), whereas, in vaccinated participants, anxiety levels were minimal (56.9%). A statistically significant correlation was discovered when comparing the scores of the vaccinated and non-vaccinated individuals as well as when comparing the professions of vaccinated participants, whereas no association was found with the gender and education level of participants. Vaccination is necessary for all entitled individuals to control the spread of coronavirus. It was discovered that there was an increase in anxiety levels before the vaccination was introduced. The anxiousness was greatly lessened following mass immunizations. Our research will help to raise public awareness of stigmatized mental health disorders in the healthcare industry.