A new series of chromeno-annulated cis-fused pyrano[3,4-c]pyran derivatives have been synthesized by intramolecular [4 + 2] domino Knoevenagel–hetero-Diels–Alder reactions of 1-oxa-1,3-butadienes derived in situ from 1,3-dicarbonyls/active methylenes and 7-O-prenyl derivatives of 8-formyl-2,3-disubstituted chromenones in the presence of 20 mol% ethylenediamine diacetate (EDDA) in acetonitrile under reflux conditions in good to excellent yields. The structures were established based on spectroscopic data, and were further confirmed by X-ray diffraction analysis. These compounds were evaluated for their anti-proliferative activity using an in vitro MTT cytotoxicity assay. The results clearly demonstrated that compounds 4a, 4b, 4c, 4j, 4k, 4l, 4m and 4n exhibited significant antiproliferative activity against human A549 lung cancer and non-cancer MRC-5 cell lines along with potent inhibitory activity against human neuroblastoma SK-N-SH cancer cell lines. Among these, compounds 4a, 4b and 4j displayed the most potent anti-proliferative activity against human lung cancer A549 cell lines, while 4a and 4b displayed against neuroblastoma SK-N-SH cancer cell lines when compared to the standard doxorubicin.