Marine Protected Areas (MPAs) are essential for conserving marine biodiversity and ensuring the sustainability of marine ecosystems. This study examines the current management and monitoring practices within the Mediterranean Sea. Utilizing a questionnaire distributed to 93 MPA managers, we received 29 complete responses, providing insights into the governance, management plans, and monitoring strategies employed across the region. Our findings reveal significant variability in the implementation of management plans, with 48% of respondents reporting full implementation and 41% employing ad hoc monitoring strategies. Traditional monitoring methods, such as visual censuses, remain prevalent, while 28% of respondents reported using eDNA metabarcoding as an additional tool. The integration of eDNA metabarcoding has shown substantial potential in enhancing the accuracy and efficiency of biodiversity monitoring, particularly in detecting species diversity and invasive species. Despite these advancements, challenges persist, including funding constraints, lack of human resources, and inadequate data-sharing practices. To address these issues, we recommend increasing funding, standardizing monitoring protocols, enhancing regional cooperation, and promoting adaptive management informed by robust monitoring data. This study underscores the transformative potential of eDNA metabarcoding in MPA management and highlights the need for strategic improvements to ensure the sustainability and ecological health of Mediterranean MPAs.