Pleurotus eryngii (king oyster mushroom) possesses potent larvicidal effectiveness against certain mosquito vectors. The mushroom extract is rich in certain polysaccharides and terpenoids that can confer such larvicidal activity. In this study, we present a simple protocol for P. eryngii mushroom extract isolation, characterization, and assessing its efficacy in Anopheles culicifacies, a significant malaria vector in South Asia. Methanolic and acetonic extracts of P. eryngii significantly caused mortality in third-instar and fourth-instar larvae. Bioassays determined lethal concentrations (LC50 and LC90) at different hours. The methanolic extract was exceptionally effective, resulting in 100% mortality within 48 h against both instars at 500 ppm. The acetonic extract exhibited larvicidal performance (85% mortality) for 5–48 h. GC-MS (Gas chromatography-mass spectrometry) analysis revealed unique bioactive compounds, such as unique polysaccharides that could be associated with the observed effects. These findings indicate the potential utility of P. eryngii extracts as part of an environmentally compatible strategy for vector control, particularly in malaria-endemic areas.