A series of 3-diarylethyne quinazolinone compounds (4a–ad) were synthesized and evaluated for their cytotoxicity. They exhibited significant anticancer activity against MDA-MB-231, A549, MIAPaCa-2 and Colo-205 cancer cell lines and were highly effective in the MCF-7 cancer cell line with IC50 values ranging from 2.27 to 3.88 mM. FACS analyses and BrdU assays revealed that there is G1 cell cycle arrest. Moreover, cells treated with these compounds show up-regulation of p53, p21, p16, TRF1 and POT1 and down-regulation of SKP2, TRF2 and tankyrase protein levels. To assess the senescence inducing ability of these compounds further, a senescence associated b-gal assay was performed. Interestingly, two of these compounds (4s and 4w) exhibited significant telomerase inhibition activity and inhibited tankyrase-1 protein, a protein partner associated with the sheltrin complexes of telomeres.