Staphylococcus aureus is a leading cause of chronic bovine intramammary infections (IMI) resulting in major economic losses for the dairy industry. Most antibiotics used by milk producers to treat S. aureus IMI have limited efficacy. To find novel control measures for S. aureus, we need to identify new cellular targets. Genes and proteins involved in virulence or that are essential for the pathogen growth in vivo represent a large pool of yet unexploited targets for vaccine and antibiotic discoveries. For such a purpose, we studied the transcriptome of S. aureus during the infection process. Mammary gland quarters were experimentally infected by a variety of S. aureus strains and milk and bacteria were recovered at each milking over 18 d. The genes expressed in bacteria grown in vivo were identified by hybridization of transcripts to DNA arrays and results were confirmed by qPCR. Several genes were found to be commonly expressed by S. aureus strains during infection. Genes of interest were selected for mutagenesis. Attenuation of gene-disrupted S. aureus mutants during experimental IMI in cows validated the role of such genes in the infection process. Proteins derived from selected genes were used for immunization of cows before an experimental challenge with S. aureus. Vaccinated cows better handled the experimental infection (SCC, bacterial count in milk, milk quality, and milk production). Such a vaccine is now under development. Besides, another in vivo-expressed transcript represented an interesting target for antibiotic action. This novel drug target is a non-coding RNA (guanine riboswitch) which is completely different from protein targets of traditional antibiotics. We subsequently identified small drug-like molecules that mimic the natural riboswitch ligand and prevent expression of an essential gene (guaA). We further showed antibiotic activity of one such artificial ligand during treatment of experimental S. aureus IMIs in cows. Transcriptomic is a reliable approach for the development of novel treatment and control measures for bovine mastitis.