Background Healthcare providers are facing an increasing threat as a result of confronting COVID-19. The aim of the present study was to assess the changes in compliance with the protective behavioral recommendations after taking the first round of COVID-19 vaccine among healthcare workers in Southern Ethiopia. Methods A cross-sectional study design was used in which quantitative data were collected using a survey method. A self-administered questionnaire was distributed to a sample of healthcare workers who were selected through a multi-stage cluster sampling technique. From the 403 healthcare workers who participated in the survey, 236 properly completed and returned the questionnaires. After inserting it into SPSS software, data were analyzed using frequency tables, percentage distributions, and logistic regression coefficients. Results While 30.5% of the healthcare workers disclosed a decreasing experience of wearing mask, 30.1% revealed that their experience of regularly washing hands has decreased after taking the first round of COVID-19 vaccine. The main reasons for not regularly wearing a mask are its inconvenience (60%), the need to appear indifferent (38.5%), and that they cannot afford to buy one due to its cost (15.4%). Above all, respondents’ level of compliance with regular wearing of a mask is significantly associated with sex (OR = 3.165, P<0.05; 95% CI: 1.186–8.448), the type of organization in which they are currently working (OR = 3.553, P<0.05; 95% CI: 1.185–10.647), knowing someone ever infected by COVID-19 (OR = 0.091, P<0.001; 95% CI: 0.030–0.275), and the belief that COVID-19 causes a severe illness (OR = 0.249, P<0.05; 95% CI: 0.299–2.615). Conclusion We have found significant reductions in adherence to the usual protective mechanisms. Therefore, increased access to the personal protective materials, including water, should be created and the healthcare workers should be continuously informed about the serious consequences of ignorance of self-protective behavior.