How can one identify the antibiotic compounds in a complex extract without prior separation? The modern workflow for the functional identification and characterization of bioactive compounds for medicinal or agrochemical purposes is shown featuring a new “reverse metabolomics” method. We start with fungal fruiting bodies, a neglected source of natural products. The process chain with the collection of mushrooms, followed by their extraction, antibiotic and metabolic profiling by mass spectrometry and thereupon based property guided isolation, and eventually structure elucidation spectroscopic methods. The identification of the relevant peaks of the crude extracts thereby is aided by a “reverse metabolomics” process newly developed in our group. It uses algorithms and modern computational methods for an activity-correlation-analysis (AcorA) linking spectroscopic data from metabolic profiles of the fungal extracts to biological activity profiles of the same samples. This approach leads to a more efficient and faster recognition of novel compounds ready for further development toward their environmentally friendly application as drugs or agrochemicals.