Infections caused by Gram-positive bacterial pathogens such as Staphylococcus aureus (S. aureus), often treated with peptidoglycan cell wall synthesis inhibitors (e.g., methicillin), represent a growing threat, and new antibiotic targets are needed for its successful treatment.1 Inhibition of the Sulfur Mobilization Factor (SUF) pathway of many Gram-positive pathogenic bacteria, the non-redundant means by which iron-sulfur (Fe-S) cluster cofactors are synthesized, represents a promising target for the development of a new class of antibiotics. Among several other auxiliary proteins, the SUF pathway of S. aureus contains an essential pyridoxal-5’-phosphate (PLP)-dependent cysteine desulfurase (SaSufS), a sulfur transferase (SaSufU), and an Fe-S cluster scaffold complex (SaSufBCD). To facilitate the development of specific and selective small-molecule inhibitors of SaSufS, a spectrophotometric assay based upon the accumulation of long-lived absorbing intermediates during the catalytic cycle of the enzyme was developed.2 Using this method, SaSufS was kinetically characterized in the presence and absence of a reducing agent (tris (2-carboxyethyl) phosphine), and the cysteine desulfurase inhibitory efficacies of both enantiomers of the well-known PLP-binding drug, cycloserine (Seromycin), against SaSufS were assessed. This method was used to assess the inhibitory potential of a series of novel inhibitors of SaSufS. Finally, methicillin-resistant S. aureus strain LAC was grown in the presence of these inhibitors, and they were found to inhibit the growth of S. aureus. Combination of these data, provide mechanistic insight into the sulfur mobilization process of the essential S. aureus SUF Fe-S biosynthetic pathway and reveal a new means of targeting both SaSufS and the cysteine desulferases of other SUF-dependent pathogens.