: This study aimed to evaluate the chemical composition and anticancer activity of Hamadan propolis essential oil and methanolic extract on HCT116 cell line. The anticancer activity of propolis methanolic extract and essential oil was evaluated by using the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) test on the colorectal HCT116 cell line. The essential oil of Hamadan propolis was analyzed by gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). The total flavonoid and phenolic content of methanolic extract were assessed using the aluminum chloride colorimetric procedures and Folin-Ciocalteu (F-C) assay, respectively. The results of MTT assay confirmed the anticancer properties of Hamadan propolis essential oil and methanolic extract on the HCT116 cell line. Moreover, 30 compounds were characterized from Hamadan propolis essential oil, the main compounds of which included β-udesmol (25.7%), α-udesmol (20.4%), α-copaen-11-ol (13.7%), and γ-udesmol (10.1%). In addition, the total flavonoid content and total phenolic content of the methanolic extract were 14.12 mg/g and 32.01 mg/g dry weight, respectively. In summary, the most important constituents of Hamadan propolis essential oil were β-u desmol, α-eudesmol, α-copaen, and γ-u desmol. Despite the cytotoxic effects of Hamadan propolis methanolic extract and essential oil on HCT116, further evaluation of anticancer effects of the most important constituents of propolis methanolic extract and essential oil is recommended.