Technical Abstract: Columnaris disease, enteric septicemia of catfish, and streptococcosis are common bacterial diseases of certain freshwater fish and are caused by Flavobacterium columnare, Edwardsiella ictaluri, and Streptococcus iniae, respectively. Various plants were selected to evaluate, isolate, and identify compounds with toxicity against these bacteria. A promising extract from the aerial parts of the terrestrial plant Atraphaxis laetevirens (Ledeb.) Jaub. et Spach (Polygonaceae Juss.) was selected for bioassay-guided fractionation using a rapid microplate bioassay. The active dichloromethane extract underwent liquid-liquid partitioning, and active fractions were further separated by normal phase column chromatography and normal phase HPLC. Nepodin and emodin were isolated from two fractions with strong toxicities against S. iniae. A chloroform fraction was further separated by normal phase column chromatography to yield two active fractions against F. columnare, and these fractions contained chrysophanol, physcion, and nepodin. Chrysophanol had strong activity and nepodin had moderate activity against F. columnare while physcion and emodin were not toxic at the concentrations tested.