Staphylococcus aureus is a Gram-positive opportunistic pathogenic bacterium responsible for a broad spectrum of diseases, ranging from mild skin infections to life-threatening septicemia. Its remarkable capacity to withstand diverse and hostile environments, including antibiotic pressure and host immune defenses, poses major challenges for effective treatment. This adaptability stems from complex regulatory networks operating at multiple levels to fine-tune the expression of virulence factors and stress response pathways. Among these mechanisms, the role of post-transcriptional RNA modifications in ribosomal (rRNA) and transfer RNA (tRNA) remains largely underexplored, despite their potential impact on translation and global gene expression. These chemical alterations (e.g. methylation, deamination, isomerization) are introduced by dedicated enzymes, many of which respond to environmental cues, thereby modulating translation efficiency and fidelity and contributing to stress adaptation. The present data set provides LC-MS/MS-based quantification of RNA modification in S. aureus across diverse RNA samples.