In the present study, ethanol extracts of 90 wild mushroom samples from Nepal, and the pure compound hispidin, were screened for their ability to inhibit β-hexosaminidase release (BHR) from rat basophilic leukemia-2H3 cells. Simultaneously, the toxicity of the extracts toward the cells was also determined, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Samples belonging to the groups Hymenochaetales and Polyporales showed promising anti-allergic activity, with Phellinus adamantinus and Ganoderma lingzhi 3 allowing a mere 19.4% and 16.7% BHR, respectively, without any cell cytotoxicity. Moreover, the 50% inhibitory concentration (IC50) values for Inonotus clemensiae and P. adamantinus were determined to be 51.24 and 50.65 μg/mL, respectively; whereas hispidin, the major bioactive compound in I. clemensiae showed an IC50 value of 82.47 μg/mL. These findings are crucial in underscoring the medicinal value of the wild mushrooms of Nepal, as a source of strong antiallergic agents.