Staphylococcus aureus is a leading cause of bacteria-associated mortality worldwide. This is largely because infection sites are often difficult to localize and the bacteria forms biofilms which are not effectively cleared using classical antibiotics. Therefore, there is a need for new tools to both image and treat S. aureus infections. We previously identified a group of S. aureus serine hydrolases known as fluorophosphonate-binding hydrolases (Fphs), which regulate aspects of virulence and lipid metabolism. However, because their structures are similar and their functions overlap, it remains challenging to distinguish the specific roles of individual members of this family. In this study, we applied a high-throughput screening approach using a library of covalent electrophiles to identify inhibitors for FphB, FphE, and FphH. We identified inhibitors that irreversibly bind to the active-site serine residue of each enzyme with high potency and selectivity without requiring extensive medicinal chemistry optimization. Structural and biochemical analysis identified novel binding modes for several of the inhibitors. Selective inhibitors of FphH impaired both bacterial growth and biofilm formation while Inhibitors of FphB and FphE had no impact on cell growth and only limited impact on biofilm formation. These results suggest that all three hydrolases likely play functional, but non-equivalent roles in biofilm formation and FphH is a potential target for development of therapeutics that have both antibiotic and anti-biofilm activity. Overall, we demonstrate that focused covalent fragment screening can be used to rapidly identify highly potent and selective electrophiles targeting bacterial serine hydrolases. This approach could be applied to other classes of lipid hydrolases in diverse pathogens or higher eukaryotes.