Mushrooms are widely-consumed fungi which contain natural compounds that can be used both for their nutritive and medicinal properties, i.e., taking advantage of their antimicrobial, antiviral, antitumor, anti-allergic, immunomodulation, anti-inflammatory, anti-atherogenic, hypoglycemic, hepatoprotective and antioxidant effects. Currently, scientific interest in natural compounds extracted from the fungal species is increasing because these compounds are also known to have pharmacological/biological activity. Unfortunately, however, their mechanisms of action are often unknown, not well understood or have not been investigated in their entirety. Given the poly-pharmacological properties of bioactive fungal compounds, it was decided to carry out a multi-targeted approach to predict possible interactions occurring among bioactive natural fungal extracts and several macromolecular targets that are therapeutically interesting, i.e., proteins, enzymes and nucleic acids. A chemical database of compounds extracted from both edible and no-edible mushrooms was created. This database was virtually screened against 43 macromolecular targets downloaded from the Protein Data Bank website. The aim of this work is to provide a molecular description of the main interactions involving ligand/multi-target recognition in order to understand the polypharmacological profile of the most interesting fungal extracts and to suggest a design strategy of new multi-target agents.