A library of pyridine-based 1,2,4-triazolo-tethered indole conjugates were designed, synthesized, and evaluated for anti-proliferative activity against a panel of six human cancer cell lines. All the synthesized conjugates (<b>14a</b>-<b>q</b>) were found to be effective against the HT-29 cell line. Particularly conjugates <b>14a</b>, <b>14n</b>, and <b>14q</b> exhibited promising cytotoxicity, with IC<sub>50</sub> values of 1 μM, 2.4 μM, and 3.6 μM, respectively, compared to the standard 5-fluorouracil (IC<sub>50</sub> = 5.31 μM). Cell cycle arrest at the G0/G1 phase was observed with these compounds, the mitochondrial membrane potential was interrupted, and the total ROS production was enhanced. Western blot and immunofluorescence experiments illustrated that these compounds inhibit the expression of markers that are involved in β-catenin and PI3K pathways. Molecular dynamics simulations demonstrated that compound <b>14a</b> has major hydrophobic interactions and few H-bonding interactions with both PI3K and tankyrase proteins.