Plusbacin A(3) is a depsipeptide antibiotic isolated from Pseudomonas sp. Although the stereochemistry at the lactone stereocenter had not been determined, biological evaluation of this compound demonstrated it to have promising antibacterial activity against vancomycin-resistant enterococci. Its mechanism of action remains to be conclusively established, but it is believed to exert its antibiotic effect through inhibition of bacterial cell wall biosynthesis. In this paper, we describe the first total synthesis of plusbacin A(3) and assign the stereochemistry for the remaining unassigned lactone stereocenter.