Pimaricin (natamycin) is a small polyene macrolideantibiotic used worldwide. This efficient antimycotic andantiprotozoal agent, produced by several soil bacterial speciesof the genusStreptomyces, has found application in humantherapy, in the food and beverage industries and as pesticide.It displays a broad spectrum of activity, targeting ergosterolbut bearing a particular mode of action different to other poly-ene macrolides. The biosynthesis of this only antifungal agentwith a GRAS status has been thoroughly studied, which haspermitted the manipulation of producers to engineer the bio-synthetic gene clusters in order to generate several analogues.Regulation of its production has been largely unveiled, con-stituting a model for other polyenes and setting the leads foroptimizing the production of these valuable compounds. Thisreview describes and discusses the molecular genetics, uses,mode of action, analogue generation, regulation and strategiesfor increasing pimaricin production yields.