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. During the process of evaluating several species of plants to 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 was subjected to liquid-liquid partitioning, and active fractions were further separated by normal-phase column chromatography and normal-phase high-performance liquid chromatography (HPLC). Nepodin (3) and emodin (4) 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 (1), physcion (2), and nepodin (3). Compound 1 had strong activity, and compound 3 had moderate activity against F. columnare , while compounds 2 and 4 were not toxic at the concentrations tested.