The biodegradation of caffeine, theobromine and theophylline by wood destroying fungi Coniophora puteana were analyzed in the present study. Aquatic solution of caffeine (1 g·L−1) was performed and fungal hyphae were put into the solutions for 28 days. Vials with the fungi were stored in a biological incubator at 22 ± 2 °C for 28 days. The same design was used for vials with the fungi without the tested substances and the pure distilled water as a reference. The results of biological tests were evaluated by a liquid chromatography-mass spectrometry method. The fungal samples were analyzed each 7 days. Caffeine concentration increased in the fungal mass during the first 14 days. After that its concentration decreased. Caffeine was also degraded to theophylline and theobromine. Theophylline and theobromine concentrations rapidly increased during the first 7 days and then rapidly decreased fast to zero. The decreasing levels of all substances in fungi indicated their biodegradation during time.