Abstract The goal of this study was to analyze the physical and physicochemical composition and microbiological properties of mechanically separated meat (MSM) from Brazilian catfish (Brachyplatystoma vaillantii) and to prepare fish flour from this byproduct while assessing its technological properties, physicochemical and microscopic characteristics. After filleting, the residue was transported and stored at −22 °C and then subjected to mechanical meat separation so the fish flour could be prepared. The MSM analysis identified 17 fatty acids, of which the main ones were palmitic, oleic, stearic, palmitoleic, myristic, EPA, and DHA. The catfish MSM's amino acid profile had mainly glutamic and aspartic acids, arginine, lysine, and leucine. Salmonella spp or sulphite-reducing clostridia were not found in the MSM or the fish flour, while counts of coliforms at 45 °C (<3.0 MPN/g) and coagulase-positive staphylococcus (<1 × 101 CFU/g) were within the limits set by the Brazilian legislation. The fish residue had 78.36 g/100 g moisture, 9.52 g/100 g proteins, 10.80 g/100 g lipids and 18.41 mg TVB-N/100 g (within the Brazilian legislation standards). The best condition to prepare the fish flour was defined as 60 °C due to the best fit to the experimental data. The results suggest that using residues from Brazilian catfish filleting to make fish flour resulted in a product with high nutritional value able to increase value of several foodstuffs, besides contributing to the reduction of waste production in the region's fish industries.