Biosecurity is the cornerstone of prevention strategies to limit infectious pathogen spread between poultry farms. However, poor compliance of biosecurity measures is often observed when entering or exiting barns. The lack of knowledge, understanding, risk perception or technical skill are often described for anteroom-related biosecurity errors (i.e., not respecting anteroom zoning in relation to changing boots, clothing, and hand sanitation). In this study, we designed and assessed the efficacy of an intervention aiming at improving biosecurity compliance in anterooms, using substances glowing under ultraviolet light, which mimic microbiological contamination. Training exercises offered in a single session were proposed to 145 participants with an interest in animal farming (veterinary students, agricultural students, poultry farmers and poultry farm advisors). The exercises' efficacy was assessed by measuring the change in attitude and intentions of participants about specific biosecurity measures. The participants' attitudes and intentions were quantified using a questionnaire before and after training. The change in score was assessed in a multivariate linear regression model to measure the effect of various parameters on this change. The attitudes and intentions of participants were statistically improved (p < 0.05) and were affected by the initial attitude and intention score, the gender of the participants and the status of the participant (type of student or activity in the poultry industry). The use of this exercise material, perceived as playful and engaging, could be included in the framework of biosecurity training for all stakeholders of the poultry industry., Competing Interests: Declaration of Competing Interest The author whose names are listed immediately below certify that they have no affiliation or involvement in any organization or entity with financial interest (such as honoraria, educational grants, participation in speakers’ bureaus, membership, employment, consultancies, stock ownership, or equity interest, and expert testimony of patent-licensing arrangements), or non-financial interest (such as personal of professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript., (Copyright © 2024. Published by Elsevier B.V.)