There is a growing demand for the consumption of medicinal mushrooms and their products. Ganoderma lucidum and Cordyceps militaris are two important medicinal mushrooms with significant pharmacological activities. In this study, we aim to develop a preparation of the extracts of these two mushrooms, and investigate its composition, antioxidant activities, and protective effects against oxidative damage. Liquid chromatography equipped with quadrupole time-of-flight mass spectrometry (LC-QTOF MS) was employed to analyze the chemical profiles of the mixture. A total of 94 compounds were identified as belonging to phenolic acids, flavonoids, triterpenes, and nucleosides. In addition, HPLC-DAD was used to quantify major compounds. Ferulic and cinnamic acids were predominant in the phenolic acid groups, with corresponding concentrations of 0.41 and 0.29 mg/g. The concentration of ganoderic acid A was 0.08 mg/g, and the contents of adenosine and cordycepin were 0.53 and 0.14 mg/g, respectively. The mixture could scavenge free radicals and reduce ferric ions in vitro. The preparation also showed nontoxic but proliferative effects on human fibroblast cells. In addition, it could protect fibroblasts against oxidative stress, reducing 21–22% of cell death in the presence of H2O2. These findings provide more information on the applications of medicinal mushrooms in the functional food and pharmaceutical industries.