Mushrooms or macrofungi comprise 15,000 species with visible sporocarps. Many of them are used for culinary purposes and in traditional medicine. Only a small fraction of the overall estimated fungal diversity has been explored for bioactive compounds. The aim of this study was to analyze bioactive compounds in 23 samples of less studied wild mushrooms from Croatia. Their identification was difficult just using the conventional morphological approach so, it was complemented with molecular identification using DNA barcoding. The integrated approach allowed an identification of 14 different species and two species belonging to species complexes. All samples were evaluated for polyphenolic, alkaloid, and triterpenoid compounds and their antioxidant activity was determined. Psathyrella piluliformis, stands out as a species with the highest phenolic acid and alkaloids content as well as the highest antioxidant activity. Using multivariate statistical analysis, grouping of samples from the same species grown at different localities close to each other was noticed. The results suggested that production of bioactive compounds may be under genetic influence and especially alkaloids may be good chemotaxonomic markers. Therefore, correct species identification of mushrooms should preferably be done using a combination of morphological and molecular approaches.