Currently cyanoprokaryotic algae, diatoms, haptophytes, dinoflagellates, euglenoids, and rhaphidophytes are known to produce algal toxins. A previous study by the authors reported euglenoid algae producing toxin(s) in aquaculture ponds, with confirmation based on positive fish bioassays following exposure to the isolated clonal algal cultures. Toxicity was observed in euglenoid culture isolates obtained from the pond as well as a clonal, culture collection taxon. Here we provide conclusive evidence for euglenoid toxin production, including HPLC/MS, MS/MS, and NMR analyses of a clonal (non-axenic) isolate of Euglena sanguinea grown in batch culture. Following wet chemical serial fractionation, toxic activity was identified in both the methanol and hexane extracts. These extracts were then purified using HPLC. Bioassay-guided HPLC fractionation of these two extracts demonstrated that a single class of toxic compounds, identical in mass and similar in molecular structure, was produced by this organism. The toxic compounds exhibited a maximal UV absorbance at 238nm and gave diagnostic mass peaks at 306 (MH(+)) and 288 (MH(+)-H(2)O). Unambiguous molecular structural determination was carried out by high field NMR analysis operating in 1- and 2-dimensions. Though a predominant isomer represented the bulk of the toxin, several stereo- and structural isomers were evidenced by NMR, and HPLC/MS. This compound is an alkaloid similar in structure to fire ant venom. The compound exhibits ichthyotoxic, herbicidal and anticancer activity at low ppm to ppb dosages.