Zinc and indole compounds demonstrate anti-inflammatory, antidepressant, and antioxidant activity. Edible mushrooms are good sources of these substances. Therefore, in this study, we aimed to study the accumulation, release, and absorption of zinc and indole compounds from mycelial cultures of Imleria badia species using in vitro models. Samples were analyzed using the atomic absorption spectroscopy method and the reversed-phase high-performance liquid chromatography method. The highest quantities of zinc were detected in the material grown on zinc hydrogen aspartate-enriched media (176.01 mg/100 g dry weight [d.w.]). In addition, the quantity of zinc in the control biomass was approximately 12.13 mg/100 g d.w. After passive transport, the amount of zinc was detected to be around 1.40 mg/100 g d.w., whereas after active transport with CaCo-2 cells, the quantity of zinc ranged from 0.46 mg/100 g d.w. to 12.72 mg/100 g d.w. Among the organic compounds, four indole compounds were qualitatively identified, including 5-hydroxy-l-tryptophan, melatonin, l-tryptophan, and 5-methyltryptamine. These results indicate that mushrooms and their in vitro cultures not only synthesize and accumulate these compounds, but also potentially release them into the gastrointestinal tract where they can be absorbed by the human body, which is reflected as a specific health benefit.