<p>Post-harvest losses in fisheries continue to be a significant challenge to food security, public health, and economic stability, particularly in developing countries like Bangladesh. This study investigated biochemical and microbial changes in three commercially important freshwater fish species: Tilapia (<i>Oreochromis niloticus</i>), Pangas (<i>Pangasius hypophthalmus</i>), and Pabda (<i>Ompok bimaculatus</i>) collected from the northeastern region of Bangladesh. Fish samples were obtained across the supply chain, from live harvest to local markets and supermarkets. They were analyzed for microbial prevalence, biochemical indices [pH, total volatile basic nitrogen (TVBN), free fatty acids (FFA)], and sensory quality. Results revealed consistently high prevalence of <i>Vibrio</i> spp. (90–100%) across all fish species and market types, while <i>Escherichia coli</i> and <i>Salmonella</i> spp. were more frequent in local markets than in fresh or supermarket samples, which indicates contamination from poor hygiene and handling. Biochemical analyses showed significantly elevated TVBN and FFA levels in market samples compared to live fish, though values generally remained within international acceptability limits. Sensory evaluation confirmed lower quality scores for fish from local markets relative to fresh and supermarket fish, consistent with microbial and biochemical findings. Collectively, the results show that microbial and enzymatic spoilage together drive the deterioration of fish quality in the warm and humid conditions of the region. The study points to the critical need for improved hygiene practices, robust cold chain infrastructure, and consistent quality monitoring to minimize post-harvest losses, enhance food safety, and safeguard livelihoods that rely on fisheries in Bangladesh.</p>