A series of new isoxazolyl, triazolyl and phenyl based 3-thiophen-2-yl-quinoline derivatives were synthesized adopting click chemistry approach. In addition, the synthesis of new useful synthon, (2-chloroquinolin-3-yl) (thiophen-2-yl) methanol, is reported. The obtained compounds were characterized by spectral data analysis and evaluated for their anticancer activity. All the derivatives were subjected to in vitro MTT cytotoxicity screening assay against a panel of four different human cancer cell lines, liver (HepG-2), colon (HCT-116), human cervical cancer (HeLa) and breast (MCF-7). Out of a library of 17 compounds, two compounds have been identified as potent and selective cytotoxic agents against HeLa and MCF-7 cell lines. SAR studies for such hybridized analogues were investigated and phenyl derivatives were proved to be more potent than isoxazole and triazole derivatives. Furthermore, the promising compounds were selected for in vitro inhibition of EGFR-TK and Topo II enzymes. Also, they were subjected to cell cycle arrest analysis and apoptosis assay on MCF-7 cells. Our recent finding highlights these thiophene-quinoline analogues as a promising class of compounds for further studies concerning new anticancer therapies.