Gladiolus (Gladiolus grandiflorus Andrews) is a high-valued bulbous cut flower. However, the shorter postharvest life of the gladiolus, limits its marketing and commercial value. In the present investigation, the effect of lemon grass (LG) essential oil as an antimicrobial agent was studied towards increasing the vase life of gladiolus. The results revealed that as compared to control (distilled water), treatment with a lower concentration of 5 µL L−1 LG essential oil prolonged the vase life of gladiolus up to 11 days (d). Scanning Electron Microscope (SEM) observation indicated that the sample treated with 5 µL L−1 LG essential oil showed intact vasculature, suggesting reduced microbial blockage at the stem end which was further corroborated by microbial count. Biochemical analysis suggested an increased level of total soluble sugars, carotenoid content, lower MDA accumulation, and higher activity of antioxidant enzymes in LG treated flowers. Moreover, transcripts levels of genes associated with senescence viz., GgCyP1 and GgERS1a were downregulated, while expression of GDAD1 and antioxidant genes such as GgP5C5, GgPOD 1, GgMnSOD, and GgCAT1 were upregulated in LG treated cut spikes as compared to control. Among various treatments we have concluded that, the vase life of the gladiolus cut spike was improved along with the relative fresh flower weight and diameter of flower at the lower dose of 5 µL L−1 LG oil in the vase solution. Thus, LG oil as an eco-friendly agent has the potential to extend the postharvest life of cut flowers.