Background Kaempferia galanga, commonly known as aromatic ginger, is a potential herb with high medicinal value and is used to treat many diseases like diabetes and asthma. Cancer is a group of diseases characterized by abnormal cells that divide uncontrollably and spread throughout the body. Nowadays, medicinal plants are used for their essential oil activities, including anticancerous effects. Objectives The aim of this study is to establish a method for screening in vitro oil cytotoxic effects. Materials and Methods Murashige and Skoog (MS) medium with growth regulators like benzyladenine, kinetin, indole-3-acetic acid, adenine sulfate, and naphthalene acetic acid was used for plantlet production. The in vitro regenerated plants were studied for rhizome oil extraction using Clevenger’s apparatus and gas chromatography-mass spectrometry (GC-MS) analysis, followed by their cytotoxicity study against human cervical cancer cells (HeLa) and human breast cancer cells (MCF7) by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction (MTT) assay. Results Upon analysis, it has been found that this medicinal plant contains ethyl-p-methoxycinnamate (EPMC) (56.41 ± 0.34%) as a vital compound in its rhizome oil. The validation of cytotoxicity in both cell lines was done by treatment with rhizome oil concentrations ranging from 6.25 to 100 µL, respectively. The IC50 value was found to be 44.18 ± 0.65 µL/mL in HeLa and 72.94 ± 0.26 µL/mL in MCF7 cells after 24 h of incubation. Conclusion The results of this study would be helpful for this important medicinal plant to be used by pharmaceuticals in the treatment of cancer.