In this study, the chemical composition, total phenolic content and antioxidant activity (DPPH, ORAC and FRAP assays) of A. subrufescens and P. ostreatus, cultivated with handmade and commercials supplements, were compared. Additionally, the compounds ergosterol, saccharopine, and hexitol were identified in A. subrufescens by HPLC‐MS/MS. The antioxidant compound p‐coumaric acid and dihexoses was found in both mushroom species. A. subrufescens presented higher total phenolic content (73.8 ± 0.6 mg GAE 100 g−1) and antioxidant activity than P. ostreatus (16.6 ± 0.5 mg GAE 100 g−1). The handmade supplement based on the waste of noble grains presented statistically similar phenolic content to the mushrooms cultivated with commercial ones Spawn Mate II SE (86.1 ± 1.4 and 92.9 ± 0.3 mg GAE 100 g−1, respectively). Therefore, the results support the use of handmade supplements based on agro‐wastes as a viable alternative to the use of high‐cost commercial ones. Pleurotus ostreatus and Agaricus subrufescens supplementation (both with commercial and handmade ones) was evaluated by the antioxidant activities and chemical composition.