<p>Physiological adaptation to sustain the freezing climate is an important response featured by the plants growing in cold regions. Current research was designed to investigate the phytochemical properties of Lali Guras (<i>Rhododendron arboreum</i> Sm.) leaves altered due to freezing stress at Sandakphu (the highest peak of West Bengal). Comparative phytochemical screenings and in vitro bioactivity assays (antioxidant, antidiabetic and anti-inflammatory) were carried out to investigate the changes that&#xa0;occurred in Lali Guras leaves to sustain the extreme cold temperature of Himalaya’s frigid winter. The reduction of antioxidant and anti-inflammatory activities in vitro assays in freezing stressed leaves corresponded to the result of total phenolics. However, antidiabetic activity was found to be elevated in freezing damaged leaves. Further, GC–MS analysis was conducted to ascertain the changes in the metabolite profiles and to identify the signature molecules or markers involved in this process of adaptation. A significant increase in the peak areas of the few metabolites was noticed in correlation with the result of fatty acid estimation. The exclusive detection of stress induced fatty acids such as 9,12,15-octadecatrienoic acid, 2,3-dihydroxypropyl ester, (Z,Z,Z)-, and 6,9,12,15-docosatetraenoic acid, and methyl ester in freezing damaged leaf sample suggested that the unsaturated fatty acid biosynthesis pathways were activated as a response strategy to withstand the freezing stress. Moreover, this research presented the proposed biosynthesis pathways and highlighted the enzymatic activities responsible for phytochemical alterations such as switch on, switch off, upregulation and downregulation of various biomolecules.</p>