Old yellow enzymes (OYEs) are pervasive in microbial systems, yet their impact on microbial stress response particularly during infection situations, remains elusive. In the genome of the gram-positive opportunistic pathogen, Staphylococcus aureus (S. aureus), two paralogous genes (ofrA and ofrB) encode for two OYEs. We investigated the function and the regulatory pathway of the highly conserved ofrA in S. aureus employing transcriptional, functional, and genetic analysis. Our findings reveal that OfrA is integral to S. aureus survival in human blood and intra-macrophages. Furthermore, ofrA mutation renders S. aureus more sensitive against reactive electrophilic, oxygen, and chlorine species (RES, ROS, and RCS). Additionally, ofrA responds to nutrient depletion through a tightly controlled transcriptional axis. In summary, ofrA significantly contributes to S. aureus fitness in infection conditions through stress response mechanisms.