Background <p><i>Thymus daenensis</i>, an aromatic medicinal plant endemic to Iran, produces essential oils rich in thymol and carvacrol, which exhibit potent antioxidant and antimicrobial properties. Drought stress can alter secondary metabolite biosynthetic pathways, potentially enhancing the plant’s stress tolerance mechanisms. Polyamines (PAs), particularly putrescine (Put), are known to mitigate drought-induced damage by modulating stomatal conductance, enhancing osmotic adjustment, and regulating oxidative stress responses. This study investigated the effects of exogenous Put on <i>T. daenensis</i> under drought stress, with a focus on gene expression related to terpenoid biosynthesis, secondary metabolite accumulation, and free PA content.</p> Results <p>Six-week-old <i>T. daenensis</i> plants were exposed to drought stress induced by 15% (w/v) polyethylene glycol (PEG) 6000, with or without foliar application of Put. Gene expression analysis (RT-qPCR), HPLC-based quantification of free PAs, and GC–MS profiling of essential oil composition were performed. Put treatment significantly increased endogenous Put and spermidine levels in drought-stressed plants, whereas no significant changes were observed under non-stress conditions. Moreover, in drought-stressed plants, Put application upregulated <i>TPS2</i> and downregulated <i>DXR</i> expression. This transcriptional modulation was accompanied by increased levels of <i>γ</i>-terpinene and <i>p</i>-cymene, precursors of thymol and carvacrol, while the levels of thymol and carvacrol were reduced.</p> Conclusions <p>Exogenous application of putrescine modulates key genes involved in terpenoid biosynthesis and alters the essential oil composition of <i>T. daenensis</i> under drought stress, potentially enhancing its adaptive metabolic responses.</p>