The fungus Pleospora papaveracea is a potential biocontrol agent for opium poppy. The objective of this study was to characterize the growth and production of propagules of P. papaveracea on various substrates and determine their infectivity on opium poppy. Pleospora papaveracea was grown on agar media containing wheat bran, corn cobs, soy fiber, cottonseed meal, rice flour, cornstarch, pectin, dextrin, or molasses, all with the addition of brewer's yeast (BY). Maximum radial growth of P. papaveracea occurred on molasses, soy fiber, and wheat bran media. Pleospora papaveracea produced chlamydospores on dextrin–BY and cornstarch–BY only. Pleospora papaveracea growth in liquid media with 1% (wt/v) dextrin, cornstarch, soy fiber, or wheat bran resulted in the production of greater than 106 colony-forming units (cfu) ml−1 within 3 to 5 d of incubation. Pleospora papaveracea produced less than 105 chlamydospores ml−1 after 10 d of incubation in wheat bran–BY and soy fiber–BY liquid media compared with the production of greater than 105 chlamydospores ml−1 after 5 d of incubation in dextrin–BY or cornstarch–BY liquid media. Fewer cfu were produced by P. papaveracea in 0.25% dextrin or 0.25 and 0.50% soy fiber liquid media than with 1 or 2% substrate. Greater than 107 chlamydospores g−1 dry weight and 108 cfu g−1 dry weight of P. papaveracea were produced in dextrin–BY liquid media in a commercial bench-top fermentor. After air drying biomass for 6 d, propagules of P. papaveracea remained infective on opium poppy. Mycelia and chlamydospores of P. papaveracea grew and formed appressoria during the infection process. Air-dried biomass, when rehydrated in 0.001% Tween 20, caused necrosis within 48 h after application to detached opium poppy leaves. At least 94% of the propagules from air-dried biomass that germinated and infected detached opium poppy leaves were of mycelial origin.