Introduction: Frontline healthcare workers (HCWs) are at higher risk of Covid-19 infection compared to general population. Adherence to COVID-19 preventive measure is an important factor in reducing the risk of exposure to the HCWs. Thus, we conducted a study to evaluate the knowledge and attitude toward COVID-19’s preventive measure among the HCWs. Material and Methods: We performed a cross-sectional study among HCWs in COVID care center of Bharati Hospital, Sangli, between June 2021 to December 2021, A validated self-questionnaire on the knowledge of hand-washing and donning-doffing, as well as the attitude towards covid-19 appropriate behavior was administered to the HCWs. A statistical analysis was performed using a Microsoft Office 365 and SPSS-22. Results: Evaluation of knowledge on hand-washing and donning-doffing shown that majority of study participants had an average knowledge (77.12%), whereas only 7.7% had good knowledge and another 14.9% had poor knowledge. Knowledge of HCWs were significantly associated with donning-doffing training (p <0.05). Majority of study participants (94.29%) demonstrated an appropriate behavior to COVID-19. Conclusion: As the study demonstrated an average knowledge on Covid-19 appropriate behavior after receiving a training, a periodic training on infection control and prevention in a healthcare setting and orientation programs related to COVID-19 appropriate behavior is indicated.