Butenolides are well‐known signaling molecules in Gram‐positive bacteria. Here, we describe a novel class of butenolides isolated from a Gram‐negative Pseudomonas strain: the styrolides. Structure elucidation was aided by the total synthesis of styrolide A. Transposon mutagenesis enabled us to identify the styrolide biosynthetic gene cluster and using a homology search we discovered the related and so‐far unknown acaterin biosynthetic gene cluster in another Pseudomonas species. Mutagenesis, heterologous expression and identification of key shunt and intermediate products were crucial to propose a biosynthetic pathway for both Pseudomonas‐derived butenolides. Comparative transcriptomics suggests a link between styrolide formation and the bacterium’s regulatory networks.