The utilization of naturally occurring compounds as a basis for the development of promising therapeutically-active agents still attracts great interests. Unlike chemical synthesis, biotransformation provides a powerful tool for introducing complex multi-step structural modifications of substrates in a simple cost-effective manner with mild conditions. Methylxanthines and methylurates are the most abundant, consumed and biologically active purine alkaloids in nature. The microbial transformation of some natural purine alkaloids was explored using a collection of different filamentous fungi. Five substrates were selected for microbial screening, namely; caffeine (CF), theophylline (TP), theobromine (TB), theacrine (TC) and methylliberine (ML). Results revealed the capacity of a number of fungi of different genera and species to either broadly or selectively utilize substrates. Moreover, some of these fungi could efficiently ferment the substrates and produce metabolites in a relatively high yield. These metabolites may have the potential to serve as bioactive agents that can be obtained through natural eco-friendly biotransformation.