A series of new indole–tetrazole derivatives were designed and synthesized to develop potential anti-breast cancer agents. The compounds exhibited in vitro anti-proliferative activity against ER-α positive T-47D (IC₅₀ = 3.82–24.43 μM), MCF-7 (IC₅₀ = 3.08–22.65 μM), and ER-α negative MDA-MB-231 (IC₅₀ = 7.69–19.4 μM) human breast cancer cell lines. Compounds 5d and 5f displayed significant anti-proliferative activity compared to bazedoxifene (IC₅₀ = 14.23 ± 0.68 μM), with IC₅₀ values of 10.00 ± 0.59 and 3.83 ± 0.74 μM, respectively, against the ER-α dominant T-47D cell line. Also, both compounds showed non-significant cytotoxicity against normal cells HEK-293. Further, the ER-α binding affinity of 5d and 5f was assessed through a fluorescence polarization-based competitive binding assay, where 5d and 5f have shown significant binding with IC₅₀ = 5.826 and 110.6 nM, respectively, as compared to the standard drug bazedoxifene (IC₅₀ = 339.2 nM). Western blot analysis confirmed that compound 5d reduced ER-α protein expression in T-47D cells, hindering its transactivation and signalling pathways. Additionally, a molecular docking study suggests that compounds 5d and 5f bind in such a fashion that induces conformational changes in the protein, culminating in their antagonistic effect. Pharmacokinetic profiles showed that the compounds possessed drug-like properties. Furthermore, molecular dynamics simulation studies establish the dynamic stability and conformational behaviour of the ER-α protein and ligand complex of both compounds. Additionally, 5d and 5f ensure biological feasibility as per their DFT analysis through HOMO–LUMO energy gap analysis. In conclusion, compounds 5d and 5f, exhibiting significant ER-α antagonistic activity, can act as potential lead compounds for anti-breast cancer therapies.