Ergothioneine is an effective antioxidant and is abundant in species of genus Pleurotus. This research focused on developing an ergothioneine extract from P. eryngii and P. citrinopileatus under optimal extraction conditions. The hot-water and 70% ethanol extractions yielded more ergothioneine than microwave, ultrasonic, and autoclaving extractions. Hot-water extraction with optimal conditions-125 rpm at 75°C for 5 minutes-produced P. eryngii and P. citrinopileatus extracts with 0.86 and 3.73 mg ergothioneine/g dry weight, respectively. In addition to 50% of added maltodextrin, spray-dried products from mushrooms contained 23.07-16.58% carbohydrates and 18.32-21.40% protein. The microstructure of spray-dried products showed shrunken spheres with mean particle diameters of 15.82-19.94 μm. After in vitro simulated gastric and intestinal digestion, the spray-dried P. eryngii and P. citrinopileatus products contained 88% and 91% residual ergothioneine, respectively. Overall, the spray-dried products could be used as antioxidative supplements.