Mushrooms have become a prominent focus in alternative medicine and are now the subject of increasingly detailed scientific investigation. This study explores the isolation, purification, and characterization of β-D-glucan from Lycoperdon pyriforme, with particular emphasis on its wound-healing potential. The aim of this research is to isolate, purify, and characterize β-glucan compounds from mushrooms collected in their natural habitat. Gel permeation chromatography (GPC), Fourier transform infrared spectroscopy (FTIR), and nuclear magnetic resonance (NMR) spectroscopy are employed to characterize the compound. In vitro cytotoxicity is assessed using NIH 3T3 cells, and surface morphology is evaluated via electron microscopy. Spectroscopic analyses (GPC, FTIR, and NMR) confirmed the presence of β-(1 → 3) and β-(1 → 6) linkages and indicated polydispersity in the isolated β-D-glucan. Cytotoxicity assays show no toxic effect on NIH 3T3 cells, even at 1000 μg/mL. Lower concentrations (20, 100, and 500 μg/mL) promoted cell migration and wound closure in scratch assays. Hemolytic activity remained below 5%. The findings suggest that β-D-glucan from Lycoperdon pyriforme may support wound healing due to its non-toxic, hemocompatible, and coagulation-promoting properties. Future studies will focus on elucidating its mechanisms of action and evaluating clinical efficacy.