Both the soil bacterium <i>Chromobacterium vaccinii</i> and the bacterial endosymbiont <i>Candidatus</i> Burkholderia crenata of the plant <i>Ardisia crenata</i> are producers of FR900359 (FR). This cyclic depsipeptide is a potent and selective G<sub>q</sub> protein inhibitor used extensively to investigate the intracellular signaling of G protein coupled receptors (GPCRs). In this study, the metabolomes of both FR producers were investigated and compared using feature-based molecular networking (FBMN). As a result, 30 previously unknown FR derivatives were identified, one-third being unique to <i>C. vaccinii</i>. Guided by MS, a novel FR derivative, FR-6 (compound <b>1</b>), was isolated, and its structure unambiguously established. In a whole-cell biosensing assay based on detection of dynamic mass redistribution (DMR) as readout for G<sub>q</sub> inhibition, FR-6 suppressed G<sub>q</sub> signaling with micromolar potency (pIC<sub>50</sub> = 5.56). This functional activity was confirmed in radioligand binding assays (p<i>K</i><sub>i</sub> = 7.50). This work demonstrates the power of molecular networking, guiding the way to a novel G<sub>q</sub>-inhibiting FR derivative and underlining the potency of FR as a G<sub>q</sub> inhibitor.