In an effort to develop potent cyclooxygenase-1 (COX-1) inhibitors used as anticancer agent, a series of 2′,5′-dimethoxychalcones was screened to evaluate their antiplatelet effect on human washed platelets suspension. Compound 2 exhibited potent inhibition of human washed platelet aggregation induced by collagen, significantly inhibited collagen-and arachidonic acid-induced thromboxane B 2 release, and revealed inhibitory effect on COX-1 activity. Molecular docking studies showed that 1, 2, and 4 were bound in the active site of COX-1. These indicated that the antiplatelet effect of these compounds were mainly mediated through the suppression of COX-1 activity and reduced the thromboxane formation. To investigate the mechanistic action of COX-1 inhibitor enhanced the cytotoxic effect against human bladder cancer cells, NTUB1, we assessed the cytotoxic effect of 2 against NTUB1. Treatment of NTUB1 cells with various concentrations of 2 led to a concentration-dependent increase of cell death and decrease of reactive oxygen species levels. The flow-cytometric analysis showed that 2 induced a G1 phase cell cycle arrest but did not accompany an appreciable sub-G1 phase in NTUB1 cells. In addition, compound 2 increased p21 and p27 expressions and did not inhibit the expression of COX-1 in NTUB1 cells. Our results suggested that 2 enhanced cell growth inhibition or antiproliferative activity in NTUB1 cells through G1 arrest by COX-1 independent mechanism.