BACKGROUND: Healthcare staff are among the most vulnerable groups at risk of HIV infection. The present study aims to evaluate the impact of theory of planned behavior (TPB)-based educational intervention on HIV screening among healthcare staff in Firouzabad. METHODS: This quasi-experimental study was conducted on 160 healthcare staff in Firouzabad, 2024. Participants were selected using multistage sampling method (cluster and random) and randomly assigned to either the intervention or control group (n = 80 per group). The intervention group received TPB-based educational intervention in five in-person sessions, each lasting 60 min. Data were collected using a standardized questionnaire based on TPB, which was completed by both groups before and two months after the intervention. Data were analyzed using SPSS 27.0 through statistical tests, including paired t-test, independent samples t-test, Chi-squared and McNemar test. RESULTS: After the educational intervention, 72 individuals (90%) from the intervention group and 27 individuals (33.75%) from the control group intended to perform HIV screening. The McNemar test results indicated that this difference was statistically significant (P = 0.001). Furthermore, the results revealed that 64 individuals (80%) from the intervention group and 22 individuals (27.5%) from the control group performed HIV screening two months after the educational intervention, and a statistically significant difference was observed between the two groups (P = 0.001). CONCLUSION: The current study demonstrated that an educational intervention based on the Theory of Planned Behavior significantly improved the intention of undergoing HIV screening among Firouzabad healthcare professionals, in addition to the actual conduct of screening behaviors. The intervention resulted in statistical significance in terms of changes in attitudes, subjective norms, and perceived behavioral control, all of which together resulted in a noticeable rise in the participants' screening activity. These results suggest that the Theory of Planned Behavior can be a helpful construct in the creation of focused educational efforts directed at the promotion of essential preventive health behaviors, like HIV screening, among key risk occupational groups. While the results are promising in terms of the population of this study, further investigations are needed in order to determine the long-term durability of the acquired behaviors as well as the broader generalizability of the intervention in different healthcare settings.