<jats:title>Significance</jats:title> <jats:p> Bacterial secondary metabolites are of great relevance to human society and the environment. To this day, investigations of secreted metabolites focus on single compounds, compound classes, or compounds with specific bioactivities. Comparing the supernatants of <jats:italic>Streptomyces chartreusis</jats:italic> cultivated in different media, using liquid chromatography–coupled tandem MS, we detected a great diversity of highly regulated compounds surpassing genome-based expectations. Guided by molecular networking, a new polyether ionophore was identified and subsequently purified and characterized. The approach presented here provides a basis for structure analysis for molecules produced in amounts too low for standard methods of structure elucidation. Simultaneously, it facilitates the differential analysis of secreted metabolomes, providing insights into the chemical profiles under different cultivation conditions.