Background <p>Natural plant products represent one of the most productive sources of innovative lead drugs for the treatment of a wide range of ailments. <i>Dolichos kilimandischaricus</i> (Harms) ex Taub. (Fabaceae) root extracts have been traditionally used for the treatment of HPV-related cancers. The extracts from the plant have been previously shown to have antiproliferative effects on cancer cell lines. This study aimed to isolate, purify, and analyze the phytochemicals from the leaves of <i>D. kilimandischaricus</i>.</p> Methods <p>Phytochemicals in the ethanol leaf extract were separated by column chromatography on silica gel. The structures of the compounds were elucidated using spectroscopic techniques, then compared with the reported spectral data. Cytotoxicity of the phytochemicals on HL-60 and Jurkat cells was assessed by the sulphorhodamine B (SRB) assay with chlorambucil as a positive control. Docking simulations were used to further understand the binding preferences of the isolated compounds.</p> Results <p>The isolated compounds were identified as 3<i>β</i>-stigmasterol (1) and <i>α</i>-spinasterol (2). The results showed that both compounds were more potent against HL-60 cells than Jurkat cells, with IC₅₀ values of 9.49 and 8.66&#xa0;µg/mL, respectively. The two compounds function as 1HJC and 6VG8 inhibitors, according to the docking results, with the highest negative molecular binding affinities shown for both ligands against 1HJC.</p> Conclusions <p>Therefore, this study has identified some of the phytochemicals that may be responsible for the antiproliferative activity in <i>D. kilimandischaricus</i>. These phytochemicals may provide leads in the development of compounds for treating cancer and related neoplastic diseases.</p> Clinical trial number <p>Not applicable.</p>