We have analyzed the abundance of bacterial sulfonosphingolipids, including rosette-inducing factors (RIFs), in seven bacterial prey strains by using high-resolution tandem mass spectrometry (HRMS 2 ) and molecular networking (MN) within the Global Natural Product Social Molecular Networking (GNPS) web platform. Six sulfonosphingolipids resembling RIFs were isolated and their structures were elucidated based on comparative MS and NMR studies. Here, we also report the first total synthesis of two RIF-2 diastereomers and one congener in 15 and eight synthetic steps, respectively. For the total synthesis of RIF-2 congeners, we employed a decarboxylative cross-coupling reaction to synthesize the necessary branched α-hydroxy fatty acids, and the Garner-aldehyde approach to generate the capnine base carrying three stereogenic centers. Bioactivity studies in the choanoflagellate Salpingoeca rosetta revealed that the rosette inducing activity of RIFs is inhibited dose dependently by the co-occurring sulfonosphingolipid sulfobacins D and F and that activity of RIFs is specific for isolates obtained from Algoriphagus.