C4-phenylthio β-lactams are a new family of antibacterial agents that have activity against two phylogenetically distant bacteria - Mycobacterium tuberculosis (Mtb) and Moraxella catarrhalis (M. cat). These compounds are effective against β-lactamase producing Mtb and M. cat unlike the clinically relevant β-lactam antibiotics. The structure-activity relationship for the C4 phenylthio β-lactams has not yet been completely defined. Earlier efforts in our laboratories established that the C4-phenylthio substituent is essential for antimicrobial activity, while the N1 carbamyl substituent plays a more subtle role. In this present study, we investigated the role that the stereochemistry at C4 plays in these compounds' antibacterial activity. This was achieved by synthesizing and testing the antimicrobial activity of diastereomers with a chiral carbamyl group at N1. Our findings indicate that a strict stereochemistry for the C4-phenylthio β-lactams is not required to obtain optimal anti-Mtb and anti-M. cat activity. Furthermore, the structure-bioactivity profiles more closely relate to the electronic requirement of the phenylthiogroup. In addition, the MICs of Mtb are sensitive to growth medium composition. Select compounds showed activity against non-replicating and multi-drug resistant Mtb.