A critical need exists for new biological pest management techniques for the pickleworm, Diaphania nitidalis (Stoll) (Lepidoptera: Pyralidae), a major pest of Cucurbitaceae. One potential management strategy for this pest is the use of entomopathogenic viruses. In this study, several viruses were evaluated for efficacy against pickleworm cell lines and second-stage larvae. First, selected viruses were bioassayed in vitro against one suspended cell line (IPLB-DnEs1) and one attached cell line (IPLB-DnEa3) that we derived from pickleworm embryos. In these experiments, a multiply-embedded nucleopolyhedrovirus (AgMNPV) from velvetbean caterpillar, Anticarsia gemmatalis Hübner, showed the greatest activity toward the pickleworm cell lines. Following these results, 14 entomopathogenic viruses in the families Baculoviridae, Reoviridae, and Iridoviridae were evaluated in vivo in bioassays of second instar D. nitdalis. Among these 14 viruses, only six baculoviruses showed significant activity against pickleworm larvae. AgMNPV and a multiply-embedded nucleopolyhedrovirus (AcMNPV) from alfalfa looper, Autographa californica (Speyer), were the most efficacious against second instar D. nitidalis. Moreover, five of six stilbene fluorescent brighteners significantly increased efficacy of AgMNPV against pickleworm larvae.