Colorectal cancer is strongly associated with chronic inflammation, in which cyclooxygenase-2 (COX-2) plays a pivotal role in tumor progression, making it an important therapeutic target. In this study, the antiproliferative activity of 1,4-naphthoquinones ( 3 - 18 ) in SW480 cells was investigated, and selected derivatives were characterized using computational and in vitro approaches. Initial antiproliferative screening identified compounds 3 , 8 , and 12 as the most potent candidates, prompting further investigation into their molecular mechanisms. These compounds were further assessed for their predicted interactions with COX-2 using AutoDock Vina and molecular dynamics (MD) simulations, followed by in vitro COX-2 inhibition and cell-based cytokine assays. Hoechst 33342, JC-1 staining, cell cycle analysis, and apoptosis assays were used to evaluate cellular responses. Through integrated computational and experimental approaches, we demonstrated that three compounds bind favorably within the COX-2 active site with key residues in a manner comparable to that of rofecoxib. Consistent with their ability to target COX-2, compounds 3 , 8 , and 12 showed potent in vitro COX-2 inhibitory at nanomolar (IC ₅₀ values of 10–18 nM) and modulated LPS-induced inflammatory responses, significantly reducing TNF-α, IL-8, and IL-10 release. The compounds were also associated with mitochondrial dysfunction, cell cycle changes, and apoptosis, with compound 12 emerging as the most active derivative and showing the strongest antiproliferative and pro-apoptotic effects. Our findings establish compound 12 as a promising lead candidate for the development of new anti-colorectal cancer agents and provide mechanistic insights into the interplay between COX-2 inhibition, inflammatory modulation, and apoptosis induction in inflammation-associated colorectal carcinogenesis.