The importance of doctors’ knowledge and awareness of infectious diseases was felt worldwide during the COVID-19 pandemic. With this study, we aimed to evaluate the effect of the long and dynamic pandemic process on resident physicians’ knowledge and protective behaviors for infection control in a tertiary hospital setting and protective behaviors for infection control in a tertiary hospital setting. The population of this cross-sectional study consisted of assistant physicians working at Suleyman Demirel University Faculty of Medicine Training and Research Hospital. A questionnaire evaluating information and protective practices for COVID-19 was applied to the participants through face-to-face interviews using the convenience sampling method, with an interval of one year. In the second year of the pandemic, resident physicians’ awareness of the correct use of personal protective equipment decreased (p = 0.001). Despite the continuous training, it was determined that the residents preferred masks with high protection at a lower rate when they encountered patients who received oxygen support of 5 lt/min and above (p < 0.001). To prevent the spread of COVID-19 infection in the hospital as the pandemic progresses, it has been determined that resident physicians are less prone to evaluate possible infection symptoms in patients hospitalized for non-COVID-19 reasons (p = 0.013). As a result, the data we obtained showed that despite the regular training during the pandemic and the death of many health workers, the residents’ adherence to infection control and prevention practices, which also protect them, decreased significantly in the second year of the pandemic. These valuable data showed us that good knowledge does not predict good infection control and prevention practices. Our findings show that physicians need a new education system that motivates them. In addition, psychosocial determinants, physical and mental fatigue, and institutional control factors contributing to these results and affecting individual risk perception should be recognized and prevented.