Tea tree oil (TTO) is a steam distillate ofMelaleuca alternifoliathat demonstrates broad-spectrum antibacterial activity. This study was designed to document how TTO challenge influences theStaphylococcus aureustranscriptome. Overall, bioinformatic analyses (S. aureusmicroarray meta-database) revealed that both ethanol and TTO induce related transcriptional alterations. TTO challenge led to the down-regulation of genes involved with energy-intensive transcription and translation, and altered the regulation of genes involved with heat shock (e.g.clpC,clpL,ctsR,dnaK,groES,groEL,grpEandhrcA) and cell wall metabolism (e.g.cwrA,isaA,sle1,vraSRandvraX). Inactivation of the heat shock genednaKorvraSRwhich encodes a two-component regulatory system that responds to peptidoglycan biosynthesis inhibition led to an increase in TTO susceptibility which demonstrates a protective role for these genes in theS. aureusTTO response. A gene (mmpL) encoding a putative resistance, nodulation and cell division efflux pump was also highly induced by TTO. The principal antimicrobial TTO terpene, terpinen-4-ol, altered ten genes in a transcriptional direction analogous to TTO. Collectively, this study provides additional insight into the response of a bacterial pathogen to the antimicrobial terpene mixture TTO.