In the context of structural investigation and optimization of various potential EGFR inhibitors, a novel series of asymmetrical piperazine-tethered trisubstituted thiophene-3-carboxamide selenide derivatives were synthesized and evaluated for their antiproliferative potential against selected human cancer cell lines. These derivatives, built based on a previously identified hit molecule, were synthesized via multiple-step reactions, including optimization of the C-Se cross-coupling reaction. Two compounds, 17i and 18i, displayed significant cytotoxicity (IC50 value: 4.82 ± 0.80 µM and 1.43 ± 0.08 µM) against HCT116 and A549 cancer cell lines, respectively. Quantitative analysis of apoptotic stages using Annexin V-FITC/PI double staining validated their apoptotic potential. Further, compound 18i demonstrated a remarkable inhibition of EGFR kinase, with an IC50 concentration of 42.3 nM. The lead compound 18i, with remarkable in vitro cytotoxicity, apoptosis induction capability, and EGFR inhibition, emerges as a promising candidate for anticancer therapy.