The effects of fruiting body maturity on chemical composition and antimicrobial activity of the wild mushrooms, Lactarius deliciosus and Lactarius piperatus, were evaluated. Immature and mature samples were analyzed for proximate constituents (moisture, fat, crude protein, ash, carbohydrates), nutritional value, and individual composition in fatty acids and sugars. Protein content, MUFA (monounsaturated fatty acids), and PUFA (polyunsaturated fatty acids) percentages increased with the fruiting body maturity stage, while carbohydrate and SFA (saturated fatty acids) content decreased. The maturity stage did not significantly affect the individual sugar profile. The antimicrobial activity of the mushrooms was screened against Gram positive and Gram negative bacteria and fungi, and correlated to the amounts of phenols, flavonoids, ascorbic acid, beta-carotene, and lycopene present in the immature and mature fruiting bodies. Mature fruiting bodies with mature spores presented lower antimicrobial activity, which was in agreement with the bioactive compound content found in those samples.