Freshwater farming of barramundi Lates calcarifer in Thailand is a growing sector in aquaculture, but mortalities due to infectious diseases are still a major threat to this industry. In 2018, an episode of severe mortality in juvenile barramundi was noted in a freshwater earth pond site. Fish presented with severe gill necrosis, as well as severe cutaneous hemorrhages, scale loss, and discoloration at the base of dorsal fin (saddleback lesions). Histopathology revealed extensive necrosis of skeletal muscle and gill filaments, as well as basophilic inclusion bodies and megalocytosis in muscle, gill, liver, and kidney. Scale drop disease virus (SDDV) infection was subsequently confirmed by virus-specific semi-nested PCR. Further, DNA sequences of the viral major capsid protein (MCP) and ATPase genes had a respective homology of 99.85 and 99.92% with sequences of SDDV infecting barramundi in saltwater culture. Gill necrosis and saddleback lesions are not typical lesions associated with scale drop syndrome. Their presence was explained by Flavobacterium columnare isolation from the gill, followed by positive F. columnare -specific PCR. To our knowledge, this is the first report of SDDV-associated mortality in freshwater-farmed barramundi. Furthermore, this mortality presented as a concurrent infection with SDDV and F. columnare , with typical lesions of both infections.