Based on the literature review, this study designed a questionnaire on healthcare workers' protective behaviors and perceptions of occupational infection risks to investigate the current status and influencing factors of occupational infection prevention practices among healthcare workers in Anhui Province. A cross-sectional survey was conducted from October 2024 to April 2025, involving 1 413 healthcare workers from seven hospitals across northern, southern, and central Anhui. Statistical analysis was performed using SPSS 27.0. Results indicated that the average protective behavior score among healthcare workers was 16.23±3.09, reflecting an overall moderate-to-high level of protective behavior. Regression analysis revealed that gender, department, monthly household income, perceived behavioral benefits, perceived behavioral barriers, and self-efficacy significantly influenced protective behavior (P<0.05). Significant differences in protective behavior scores were observed across groups. Self-efficacy served as the primary positive driver of protective behavior, while cognitive factors exerted a bidirectional moderating effect. The study recommends implementing differentiated protective management strategies tailored to group disparities, developing a tiered practical training system focused on enhancing self-efficacy, and addressing cognitive conflicts through targeted cognitive interventions, thereby providing a scientific basis for developing occupational infection risk countermeasures.