The choanoflagellate Salpingoeca rosetta is a microbial marine eukaryote that can switch between unicellular and multicellular states. As ope of the closest living relatives of animals, this organism has become a model for understanding how multicellularity evolved in the animal lineage. Previously our laboratories isolated and synthesized a bacterially produced sulfonolipid that induces S. rosetta to form multicellular "rosettes." In this study, we report the identification of a bacterially produced inhibitor of rosettes (IOR-1) as well as the total synthesis of this molecule and all of its stereoisomers. Our results confirm the previously noted specificity and potency of rosette-modulating molecules, expand our understanding of the complex chemical ecology between choanoflagellates and rosette-inducing bacteria, and provide a synthetic probe template for conducting further mechanistic studies on the emergence of multicellularity.