Despite their importance in cancer treatment, anticancer compounds face significant challenges due to drug resistance and low specificity, creating an urgent need for the discovery of more effective alternative. Herein, we report the synthesis of eleven 3-aminoimidazole[1,2-α]pyridine compounds (9-19) employing the one-pot Groebke-Blackburn-Bienayme three-component reaction (GBB-3CR). The cytotoxicity of the synthesised compounds was evaluated against three cancer cell lines (MCF-7, HT-29, B16F10) and a normal cell (MEF). Considering effectiveness and safety, the results demonstrated that among the eleven synthesised compounds, only compounds 12 and 14 exhibited high inhibitory activity against cancer cell lines. Compound 12 with a nitro group at the C-2 position and a p-chlorophenyl group at C-3 position, showed the highest inhibitory activity against HT-29, with an IC