Marsdenia tenacissima (Roxb.) Wight et Arn. (MT), as a traditional Chinese and Dai herbal medicine, has anti-inflammatory, antibacterial, and antitumor properties. However, most of its main active substances are aglycones, such as tenacigenin A and tenacigenin B. As the bioavailability of MT is low and its medicinal active components are challenging to synthesize, it is primarily studied by biotransformation. This study aims to produce biotransformation products rich in pungent saponins by using MT as a fermentation medium for Ganoderma lucidum (G. lucidum). Through the preliminary screening of three medicinal fungi, it was found that G. lucidum and Ophiocordyceps sinensis (O. sinensis) can generally grow in the medium for MT; hence, the efficacy of the fermentation of the two types of fungi was screened using a mouse model of lung cancer. Finally, the co-fermentation of G. lucidum and MT was selected for further investigation. Non-target metabolomics analysis was performed on the products of MT with G. lucidum co-fermentation. We identified 12 specific saponins of MT from the fermentation products, and obtained a monomeric compound, tenacigenin A, from fermentation products. Most of the tenacigenin showed a significant upward trend, through tenacigenin A and tenacigenin B levels. The results showed that the efficacy of MT improved after fermentation by G. lucidum. Furthermore, the biotransformation of C21 steroidal glycosides in MT was the central reaction in this fermentation process. In summary, this study established a systematic and comprehensive co-fermentation system and pharmacodynamic evaluation method for MT, which not only enhanced the full utilization of effective active substances in MT but also provided a methodological reference for the development of other ethnic drugs.