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Simple Summary: In poultry production, biosecurity is essential for preventing the entry and spread of pathogens in/within farms, with positive impacts on animal health and welfare. This becomes crucial in countries in which the production reaches high productive standards, such as Italy. Although the importance of implementing biosecurity measures (BMs) in poultry farms is well recognized, compliance may not be always optimal. According to the perceptions of interviewed Italian poultry farmers and advisors, biosecurity is implemented well, with different opinions between them. Compared to farmers, advisors claim a lower level of biosecurity implementation, probably because the former have limited outlooks focused on their own farms or consider some BMs not useful and/or effective, while advisors possess a more comprehensive picture of the implementation of biosecurity due to their involvement in multiple farms and productive categories. Stakeholders, mainly advisors, provided useful suggestions for encouraging farmers and workers at poultry farms to adopt BMs they remain reluctant to implement. The level of implementation of biosecurity measures (BMs), the reasons for not implementing BMs and the effectiveness of BMs were assessed according to the perceptions of stakeholders (i.e., farmers and advisors) in Italian poultry farms. For this purpose, data were collected using a questionnaire administered to advisors (n = 37) and farmers (n = 30) of conventional broiler (n = 13) and layer (n = 13), free-range broiler (n = 8) and layer (n = 10), turkey (n = 13), duck (n = 3) and breeder (n = 7) farms between April and September 2021. The frequency of the implementation of BMs was 66.97% and 81.14% according to the answers provided by the advisors and farmers, respectively, with the breeder sector showing the highest level of implementation (85.71%). "Not knowing advantages" (21.49% for advisors) and "other/specific reasons" (21.49% for advisors and 38.32% for farmers) were the most common answers regarding the lack of implementation of BMs for all poultry sectors. Only 31.09% of farmers acknowledged the effectiveness of not-implemented BMs in contrast to 61.02% of advisors, with the layers' stakeholders being the most aware. The findings of this study may be useful for identifying failures in biosecurity and failures to develop intervention strategies to fulfil the biosecurity gaps still present in Italian poultry farms. [ABSTRACT FROM AUTHOR]