Rauvolfia serpentina (L.) Benth. Ex Kurz is a greatly appreciated medicinal plant, well-known for its therapeutic benefits in traditional medicine, particularly in Ayurveda, where the roots and whole plant are used to treat a variety of ailments. However, studies focusing on R. serpentina seeds are relatively scarce. Hence, the present study provides a novel approach by analysing the seed oil of R. serpentina extracted using the supercritical-carbon dioxide-fluid-extraction (SCFE) technique. The research employed advanced analytical methods including gas-chromatography with flame ionization detector (GC-FID), gas-chromatography-tandem mass spectrometry (GC-MS/MS), and high performance thin layer chromatography (HPTLC) to characterise the chemical composition of the extracted oil. Functional moieties were evaluated by Fourier transform infrared spectroscopy (FT-IR), while proton nuclear-magnetic-resonance (¹H NMR) spectroscopy was utilised to identify the phytometabolites as well as to assess the physico-chemical parameters. The anti-microbial potential of the supercritically extracted oil was demonstrated through its activity against Klebsiella pneumoniae. The inhibitory effects on K. pneumoniae were quantified using the broth microdilution method, showing activity at both minimum inhibitory concentrations (MIC₅₀ and MIC₉₀). Furthermore, the oil was found to be non-genotoxic, as demonstrated by the Ames assay, which showed no mutagenic effects against S. typhimurium and E. coli WP2 uvrA. Since previous reports on R. serpentina seeds and their novel contribution in the field of pharmaceutics are rather limited, the present study is of utmost importance. The study may pave the way for future investigations into the therapeutic potentials of R. serpentina seeds.