We report the discovery of six new highly substituted tropolone alkaloids, rubterolones A–F, from Actinomadura sp. 5‑2, isolated from the gut of the fungus-growing termite Macrotermes natalensis. Rubterolones were identified using fungus-bacteria challenge assays and a HRMS-based dereplication strategy, and characterized by NMR and HRMS analyses and X-ray crystallography. Feeding experiments and subsequent chemical derivatisation led to a first library of rubterolones derivatives (A‒L). Genome sequencing and comparative analyses revealed their putative biosynthetic pathway, which was supported by feeding experiments. This study highlights how gut microbes can present a prolific source of secondary metabolites.