Colorectal cancer is the third most prevalent cancer in Thailand, prompting the search for alternative or preventive treatments using natural constituents. In this study, the authors employed hydrodistillation to extract Cymbopogon citratus (DC.) Stapf (lemongrass) essential oil (LEO) from plants in northeastern Thailand and assessed its chemical profile, antioxidant, antimicrobial, and anticancer properties. The LEO displayed potent antioxidant activities in DPPH and hydroxyl scavenging assays with IC50 values of 2.58 ± 0.08 and 4.05 ± 0.12 mg/mL, respectively, and demonstrated antimicrobial activities against Escherichia coli, Cutibacterium acnes, Streptococcus agalactiae, and Staphylococcus aureus at 8–10 µg/mL. At 48 h, the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay showed the LEO exhibiting low cell viability (3%) at concentrations of 200–400 µg/mL, with an IC50 value of 82.46 ± 1.73 µg/mL, while in the clonogenic assay it exhibited a lower IC50 value of 23.11 ± 1.80 µg/mL. The GC-MS analysis identified citral (79.24%) consisting of 44.52% geranial and 34.72% neral, and β-myrcene (5.56%). The addition of LEO significantly influenced apoptotic genes (Bcl-2, Bax, p21, and Caspase-3) and proteins, as indicated by real-time polymerase chain reaction (RT-PCR) and Western blot studies. Results suggested that LEO initiated apoptosis through intrinsic pathways and demonstrated potential as a chemopreventive, antimicrobial, and antioxidant agent with substantial health advantages.