Kaptai Lake (KL), the largest inland watershed in Bangladesh (ca. 700 km2) and one of Southeast Asia’s largest artificial reservoirs, features an abundant variety of indigenous fishery species. Moreover, it provides a plethora of ecological benefits to society. Nevertheless, the KL is suffering from multidimensional natural and anthropogenic stressors that threaten these wetlands’ sustainability. Though the legal framework assures sustainable conservation of fisheries resources, the implementation scenarios of fisheries laws, regulations, and policies in the KL wetland are insufficient. This study aimed at assessing the fisher’s perception of the regulation implementation efficiency of the Protection and Conservation Fish Act of 1950, while analyzing the effectiveness of the legal framework in the context of biodiversity conservation and the management sustainability of KL. By integrating qualitative and quantitative data collected through participatory Rural Appraisal (PRA) tools viz. 225 interviews with fishers, four focus group discussions, and 12 key informant interviews, the investigation was performed in four selected areas in KL. The findings show that fishers routinely disregard laws and restrictions of the Protection and Conservation of Fish Act 1950 due to various socioeconomic and political forces. Although the annual fish harvest rate from KL appears to be increasing, the lake is losing its charismatic biological diversity primarily due to inappropriate and ineffective enforcement of fishing regulations. Many fishers believe that they still follow the act’s significant laws and regulations while being involved in several destructive and prohibited fishing practices. There is a link between community awareness, the scope of the act’s provisions, the effectiveness of its enforcement, and the strength of its execution. One of the leading causes of biodiversity loss in the KL is inadequate and ineffective fishing regulations. Improvement in the enforcement of the fishing act may be the prominent option to ensure better biodiversity conservation and sustainable management of this wetland. This result calls for functional and policy attention to revising the regulations to account for socioeconomic and political elements contributing to environmental degradation. This study also highlights the urgent need for transdisciplinary collaboration initiatives and synchronous cooperation among the agencies in order to effectively implement the fishing laws and contribute to better conservation and sustainability of the Kaptai lake fisheries resources.