Infestations of fungus gnats (Diptera: Sciaridae) can reduce the production of oyster mushrooms (Pleurotus spp.) grown as food crops within controlled environments. To develop sustainable management tactics, the objectives of this study were to assess the efficacy of Bacillus thuringiensis var. israelensis (Bti) and Steinernema feltiae against fungus gnat larvae. A container bioassay was developed, whereby pasteurized straw was inoculated with Pleurotus columbinus and then treated with Bti (GnatrolÂ®), S. feltiae (NemashieldÂ®), or water. Field-collected, gravid fungus gnats (Lycoriella sp.) were released into each container for ovipositing onto the straw, thereby exposing the F1 larvae to treated or untreated substrate. Sticky cards affixed within the bioassay containers entrapped fungus gnats emerging from the substrate as an indirect measurement of larval survivorship. Following three independent bioassays, fewer fungus gnats emerged from straw treated with Bti compared to S. feltiae and the water control. Three additional bioassays conducted using straw inoculated with Pleurotus ostreatus also demonstrated that fewer fungus gnats emerged from straw treated with Bti compared to S. feltiae and the untreated control. Steinernema feltiae ranged from ineffective to slightly effective at reducing adult emergence, which could be a function of Pleurotus spp. being known to immobilize and digest nematodes. Monitoring the weight of treated and untreated substrates 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.