Most of the current cancer chemotherapeutics are associated with harsh and undesirable side effects, including toxicity and chemoresistance, driving the need for safer and more effective alternatives. In this study, the antiproliferative activities of the methanolic extract of Tetrapleura tetraptera fruits and nine different fractions (C1–C9) from the column chromatographic separation of the extract against leukemia (Jurkat) and human breast cancer (MCF-7) cell lines were investigated using a tetrazolium-based colorimetric assay. Phytochemical screening of the extract and fractions found alkaloids, carbohydrates, flavonoids, glycosides, phenols, saponins, steroids, tannins, and terpenoids in the methanolic extract. Most of the fractions exhibited antiproliferative activity (>100 μg/mL) with the Jurkat cells being more susceptible than the MCF-7 cells. Four of the collected fractions C4, C3, C5, and C2 had good selective indices in decreasing order of activity, in the case of Jurkat cells. Liquid chromatography-mass spectrometry analysis of all samples (except for C4 and C9) revealed that C1, C2, C3, and C5 each had a single component. More importantly, fractions C2, C3, and C5, which were selective to Jurkat cells, also had the same retention time of 1.846 min. Fractions C6 and C8 had two components, with C7 having four components. This study serves as a basis for further work to isolate and characterize potential anticancer agents from the fractions of extracts of T. tetraptera fruits.