Recently reported multihalogenated (CF₃/Cl) anilides of 1-hydroxynaphthalene-2-carboxanilides showed significant activity against both the reference strain Staphylococcus aureus ATCC 29213 and clinical isolates of methicillin-resistant S. aureus (MRSA). This fact inspired further investigation of the effect of these compounds on staphylococci. Chemoproteomics is a tool for investigating protein targets of potential drugs. It makes it possible to understand the effect of a bioactive molecule on a living system. An activity-based protein profiling (ABPP) method was employed using highly active, moderately active, and inactive ring-substituted 1-hydroxynaphthalene-2-carboxanilides as probes. The experiment was performed on the universally sensitive collection strain S. aureus ATCC 29213. Tryptic cleavage of proteins was performed prior to HPLC-MS/MS analysis. Protein profiles of control samples (S. aureus cells) and profiles of S. aureus treated with inactive/active derivatives were investigated and compared to each other. More than 1000 proteins were analyzed, with approximately 70% of the proteins was increased and 30% of the proteins was decreased after treatment with the investigated compounds. Under the influence of highly active compounds, the bacteria did not survive, although the expression of some life-supporting proteins (e.g., serine-aspartate repeat protein D) was observed; on the contrary, staphylococcal collagenase was one of the most inhibited enzymes. The functions of a set of proteins increased/decreased by the studied derivatives were characterized, on the basis of which the effect of bioactive agents against S. aureus can be monitored.