<jats:title>Abstract</jats:title><jats:p>Myxobacteria have proven to be highly valuable sources of natural products, as they produce a variety of secondary metabolites with unique structures and often new modes of action. In this study, high‐content screening is demonstrated to be a convenient tool for bioactivity‐guided isolation of natural products from crude bacterial extracts. By the application of focused, image‐based screens we were able to identify over 30 novel chondramide derivatives from <jats:italic>Chondromyces</jats:italic> sp. MSr9030, some of which were present in only minute amounts. These cyclic depsipeptides were shown to target actin filaments with a similar binding mode to that of the mushroom toxin phalloidin. Fermentations of the myxobacterial strain were carried out under improved cultivation conditions, and supplementation of the culture broth with potassium bromide afforded the production of brominated analogues that are superior (in terms of biological activity) to all chondramides described to date. Initial biological profiling of 11 new derivatives in comparison to the reference compounds (chondramides A–C) showed that bromo‐chondramide C3 and propionyl‐bromo‐chondramide C3 are the most active in cell‐based studies, with GI<jats:sub>50</jats:sub> values on human cancer cell lines in the low nanomolar range. Given that these brominated C3 analogues were also less potent on noncancerous human cells (by a factor of 2 to 4 in comparison to cancer cell lines), our results can aid further structure–activity relationship‐guided development of chondramides, either as molecular probes or pharmaceutical agents.