This study was to determine the co-chemotherapeutic effect of the chloroform fraction of bandotan (Ageratum conyzoides L.)(CFB) and its combination with 5-Fluorouracil (5-FU) by in vitro and in silico assay. Ethanolic extract of bandotan were fractionated with chloroform. Thin Layer Chromatography (TLC) used to identify active compound and in vitro study with MTT Assay to determine the viability of HeLa cells after extract treatment. Molecular docking used Autodock Vina for in silico study to visualize molecular interaction and affinity between nobiletin and 5-FU with Bcl-XL protein. The result of TLC for CFB showed the Rf value of 0.75, it has the similar value with quersetin standard and indicated that CFB contains flavonoid compound. The Molecular docking had ΔG for nobiletin and 5-FU were-8.0 and-4.7 kcal/mol, respectively. This result showed that the affinity of nobiletin with Bcl-XL protein higher than 5-FU. Single cytotoxic assay of CFB and 5-FU showed the IC 50 value of 30 μg/ml and 45 μg/ml, respectively. Combination assay of CFB and 5-FU showed the CI value of 0.36, meaning the presence of synergistic effects. CFB has a positive effect to inhibit viability of HeLa cervical cancer cells and potential to develop as co-chemotherapy agent with 5-FU.