In 2010, a mass mortality of pufferfish in Hawaii was dominated by Arothron hispidus showing aberrant neurological behaviors. Using pathology, toxinology, and field surveys, we implicated a series of novel, polar, marine toxins as a likely cause of this mass mortality. Our findings are striking in that 1) a marine toxin was associated with a kill of a fish species that is, itself, toxic; 2) we provide a plausible mechanism to explain clinical signs of affected fish, and 3) this epizootic likely depleted puffer populations. Whilst our data are compelling, we did not synthesize the toxin de-novo, we were unable to categorically prove that the polar toxins caused mortality or that they were metabolites of an undefined parent compound. However, our approach does provides a template for marine fish kill investigations associated with marine toxins and inherent limitations of existing methods. Our study also highlights the need for more rapid and cost-effective tools to identify new marine toxins, particularly small, highly polar molecules.