Bacterial secondary metabolites exhibit diverse remarkable bioactivities and are thus the subject of study for different applications. Recently, the individual effectiveness of tripyrrolic prodiginines and rhamnolipids against the plant-parasitic nematodeHeterodera schachtii, which causes tremendous losses in crop plants, was described. Notably, rhamnolipid production in engineeredPseudomonas putidastrains has already reached industrial implementation. However, the non-natural hydroxyl-decorated prodiginines, which are of particular interest in this study due to a previously described particularly good plant compatibility and low toxicity, are not as readily accessible. In the present study, a new effective hybrid synthetic route was established. This included the engineering of a novelP. putidastrain to provide enhanced levels of a bipyrrole precursor and an optimization of mutasynthesis, i.e., the conversion of chemically synthesized and supplemented monopyrroles to tripyrrolic compounds. Subsequent semisynthesis provided the hydroxylated prodiginine. The prodiginines caused reduced infectiousness ofH. schachtiiforArabidopsis thalianaplants resulting from impaired motility and stylet thrusting, providing the first insights on the mode of action in this context. Furthermore, the combined application with rhamnolipids was assessed for the first time and found to be more effective against nematode parasitism than the individual compounds. To obtain, for instance, 50% nematode control, it was sufficient to apply 7.8 μM hydroxylated prodiginine together with 0.7 μg/ml (~ 1.1 μM) di-rhamnolipids, which corresponded toca.¼ of the individual EC50values. In summary, a hybrid synthetic route toward a hydroxylated prodiginine was established and its effects and combinatorial activity with rhamnolipids on plant-parasitic nematodeH. schachtiiare presented, demonstrating potential application as antinematodal agents.Graphical Abstract