Infestations of fungus gnats (Diptera: Sciaridae) can reduce the production of oyster mushrooms (Pleurotus spp.) grown as food crops within controlled environments. The objectives of this study were to assess the efficacy of Bacillus thuringiensis var. israelensis (Bti) and Steinernema feltiae against fungus gnat larvae. A bioassay was developed, whereby pasteurized straw was inoculated with Pleurotus columbinus and treated with Bti (Gnatrol®), S. feltiae (Nemashield®), or water. Fungus gnats (Lycoriella sp.) were released into each bioassay container for ovipositing onto the straw, thereby exposing the F1 larvae to treated or untreated substrate. Sticky cards within the containers entrapped fungus gnats emerging from the substrate as an indicator of larval survivorship. Following three bioassays, fewer fungus gnats emerged from straw treated with Bti compared to S. feltiae and the water control. Three additional bioassays using Pleurotus ostreatus also demonstrated that fewer fungus gnats emerged from straw treated with Bti compared to S. feltiae and the untreated control. Steinernema feltiae was generally ineffective. Monitoring substrate weight in the bioassay containers over time indicated that Bti and S. feltiae did not impede colonization by P. ostreatus. Incorporating Bti into straw substrate is a promising approach for managing fungus gnats infesting Pleurotus spp.