Based on 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 medical personnel 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 efficacy, 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 emerged as the primary positive driver of protective behavior, while cognitive factors exerted a bidirectional moderating effect. Recommendations include implementing differentiated protective management strategies tailored to group disparities, prioritizing self-efficacy enhancement, establishing a tiered practical training system, resolving cognitive contradictions through targeted cognitive interventions, and providing scientific basis for developing occupational infection risk countermeasures.