This study comprehensively evaluated the effect of a Shiitake Stalk Isolated Polysaccharide (LEP) coating on Agaricus bisporus. The study findings revealed that the application of 1.5% LEP reduced weight loss, maintained firmness and chewiness, as well as the nutritional indices of mushrooms, extended the shelf life by 9 days, and maintained freshness for up to 15 days compared to the control. In addition, LEP enhanced the activities of various ROS metabolism-related enzymes such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) and significantly reduced the accumulation of hydroxides. LEP treatment during storage maintained high antioxidant activity. Additionally, LEP treatment is associated with increased activity of CCO, SDH, H⁺-ATPase, and Ca²⁺/ATPase. LEP also preserved mitochondrial membrane structural integrity, which helps to prolong shelf life. The results suggest that the LEP’s polysaccharide component may serve as a beneficial additive to prevent the deterioration of mushrooms during preservation and provide an opportunity to expand the use of LEP in edible mushroom preservation.