In this study, we analyzed the effect of media on the production of carotenoids and mycelia by using Perenniporia fraxinea. Malt extract-based medium with less than 0.1% peptone stimulated the production of carotenoids, and the one with more than 0.2% peptone inhibited its production. P. fraxinea grown in medium without malt extract did not produce carotenoids, although a small amount of peptone was added to the medium.After carotenoid production, the culture broth was separated using simple centrifugation and the supernatant was harvested as a carotenoid solution. Ethanol was used to extract carotenoids from mycelia. Carotenoid solution separated or extracted from the culture solution showed DPPH radical scavenging activity. The antioxidant carotenoids produced by P. fraxinea are derived from natural products, have no toxicity and side effects, and exhibit excellent antioxidant effects; therefore, they can be effectively used to remove oxides produced by active oxygen.