Puffballs are fungi that produce globose fruiting bodies that undergo autolysis, transforming their insides into a spore bearing, powdery mass. Mature fruiting bodies are traditionally used to treat open skin wounds. In this study, methanol extracts of two puffball species, Handkea excipuliformis and Vascellum pratense, were examined and compared in order to provide insight into the changes these mushrooms undergo during maturation, with respect to their potential use in skin care and wound treatment. Some compounds involved in skin care and regeneration were quantified, and it was found that maturation increases the concentrations of almost all of these compounds. Antioxidant activity was also more pronounced in mature fruiting body extracts, which was in correlation with the higher content of antioxidants. Tyrosinase inhibition was vastly improved with autolysis, correlating with the higher phenolic content in mature fruiting body extracts. Antimicrobial activity was negatively affected by autolysis in the case of H. excipuliformis, whereas autolysis had little effect on the antimicrobial activity of V. pratense. Autolysis generally improved the biological activity and increased the concentrations of compounds involved in skin care, which justifies the traditional use of puffballs and makes them good candidates for various potential cosmetic and medicinal skin-care products.