Alafosfalin is a phosphonodipeptide with significant activity as an antibacterial agent and as a potentiator of beta-lactam antibiotics. Alafosfalin shows good activity both in vitro and in vivo, particularly against Enterobacteriaceae. The compound acts by interfering with bacterial cell wall biosynthesis and also potentiates the activity of beta-lactam antibiotics. Alafosfalin was found to be highly active against Escherichia coli and moderately active against Serratia, Klebsiella, Enterobacter, and Citrobacter, but less active against gram-positive organisms than were beta-lactams such as cephazolin or ampicillin and inactive against indole-positive Proteus, Pseudomonas, and Acinetobacter.