Primary and secondary poisoning of nontarget wildlife with second-generation anticoagulant rodenticides has led to restrictions on their use and to increased use of first-generation anticoagulants, including diphacinone. Although first-generation anticoagulants are less potent and less persistent than second-generation compounds, their use is not without risks to nontarget species. We report the first known mortalities of threatened New Zealand lesser short-tailed bats (Mystacina tuberculata) caused by diphacinone intoxication. The mortalities occurred during a rodent control operation in Pureora Forest Park, New Zealand, during the 2008–09 Austral summer. We observed 115 lesser short-tailed bat deaths between 9 January and 6 February 2009, and it is likely that many deaths were undetected. At necropsy, adult bats showed gross and histologic hemorrhages consistent with coagulopathy, and diphacinone residues were confirmed in 10 of 12 liver samples tested. The cause of mortality of pups was diagnosed as a combination of the effects of diphacinone toxicity, exposure, and starvation. Diphacinone was also detected in two of 11 milk samples extracted from the stomachs of dead pups. Eight adults and 20 pups were moribund when found. Two adults and five pups survived to admission to a veterinary hospital. Three pups responded to treatment and were released at the roost site on 17 March 2009. The route of diphacinone ingestion by adult bats is uncertain. Direct consumption of toxic bait or consumption of poisoned arthropod prey could have occurred. We suggest that the omnivorous diet and terrestrial feeding habits of lesser short-tailed bats make them susceptible to poisoning with the bait matrix and the method of bait delivery used. We recommend the use of alternative vertebrate pesticides, bait matrices, and delivery methods in bat habitat.