Parkia javanica (Lamk.) Merr. is an ethnomedicinal leguminous plant species from northeastern India with a long history of medicinal use among various tribes of this region to treat cholera, dysentery, stomach aches, diarrhea and food poisoning, having antibacterial, wound-healing, anticancer and anti-inflammatory properties. Therefore, in this current study, the methanolic bark extract was carried out and fractionated by using flash chromatography, examined the cytotoxicity of the respective fractions on colon cancer cell lines, and evaluated the major phytochemical compounds present in the fractions using Gas Chromatography-Mass Spectrometry (GC-MS) chemical profiling. Chemical profiling of the fractions by GC-MS revealed in fraction-1 and -2, 2,4-Di-tert-butylphenol was the major compound (50.740% in fraction-1, 21.277% in fraction-2, and 7.859% in fraction-3) having reported anticancer activity. The gradation of the presence of this compound in the fractions was corroborated by the gradation of anti-colon cancer activity of the respective fractions on both the colon carcinoma cell lines. However, the presence of D-Allose in a substantial amount (20.870%) in only fraction-3 could not increase the anticancer activity of fraction-3 over the other two fractions. An in vitro cytotoxic assay guided evaluation of three flash chromatographic fractions (fraction-1, -2, and -3) of methanolic extract of Parkia javanica bark showed significant anticancer properties on two human colon carcinoma cell lines (HCT116 and SW480). The order of efficacy of the fractions was fraction-1> fraction-2 > fraction-3. In a time and dose-dependent experiment, fraction-1, being the most active one, showed an IC50 value of 16.25 µgml-1 (24 hrs), 9.94 µgml-1 (48 hrs), and 9.38 µgml-1 (72 hrs) on HCT116 and 35 µgml-1 (24 hrs), 20.14 µgml-1 (48 hrs), and 19.71 µgml-1 (72 hrs) on the SW480 cell line. Parkia javanica bark extract is bestowed with the potential of anti-colon cancer property and upon chemical profiling of different chromatographic fractions of the extract, 2,4-Di-tert-butylphenol has been identified as the primary anticancer component of the extract.