<p>Aquatic environments are subject to different pressures, often resulting in reduced ecological health. Protected areas aim to safeguard biodiversity and maintain ecological processes. In the Amazon, co-management systems in floodplain lakes have positively improved the environment and biodiversity. This study aimed to develop a biotic integrity index (IBI-FL) to assess integrity in protected and unprotected Amazonian floodplain lakes. Fish were sampled from 2018 to 2023 using gillnets and purse seines. We used a generalized linear mixed model (GLMM) to model the response of the IBI-FL to different levels of governance (formal and local). Additionally, we performed a principal component analysis (PCA) to visualize the contribution of IBI-FL metrics. We collected 68,663 fish, representing 447 species across 44 families and 14 orders from 17 lakes in four sites. The IBI-FL comprises nine metrics related to species diversity, composition, trophic category, and abundance. Results showed no significant differences in biotic integrity between formally protected sites and unprotected areas. Similarly, fully protected, preserved, managed, and open-access lakes showed comparable integrity. However, some individual lakes displayed high biotic integrity, highlighting the effectiveness of local protection. Environmental heterogeneity played an important role in the formation of biotic integrity between the lakes. The lack of clear governance effect may relate to sample size or the limited enforcement of protected area policies. We recommend expanding protected areas, increasing government investment in their maintenance, stimulating community management systems, and enhancing monitoring efforts to track changes in biotic integrity, as fundamental actions to ensure biodiversity conservation in protected areas.</p>