The bacterial burden on human health is quickly outweighing available therapeutics. Our long-term goal is the development of antimicrobials with the potential for broad-spectrum activity. We previously reported phthalazine-based inhibitors of dihydrofolate reductase (DHFR) with potent activity against Bacillus anthracis, a major component of Project BioShield. The most active molecule, named RAB1, performs well in vitro and, in a cocrystal structure, was found deep within the active site of B. anthracis DHFR. We have now examined the activity of RAB1 against a panel of bacteria relevant to human health and found broad-spectrum applicability, particularly with regard to gram-positive organisms. RAB1 was most effective against Staphylococcus aureus, including methicillin- and vancomycin-resistant (MRSA/VRSA) strains. We have determined the cocrystal structure of the wild-type and trimethoprim-resistant (Phe 98 Tyr) DHFR enzyme from S. aureus with RAB1, and we found that rotational freedom of the acryloyl linker region allows the phthalazine moiety to occupy two conformations. This freedom in placement also allows either enantiomer of RAB1 to bind to S. aureus, in contrast to the specificity of B. anthracis for the S-enantiomer. Additionally, one of the conformations of RAB1 defines a unique surface cavity that increases the strength of interaction with S. aureus. These observations provide insights into the binding capacity of S. aureus DHFR and highlight atypical features critical for future exploitation in drug development.