Pyrimido[4,5-c]quinolin-1(2H)-one derivatives were shown to exert interesting biological activities including anticancer, antimicrobial, and cardiovascular. Substitution with methoxy groups played a crucial role in the anticancer activity of known anticancer agents. This study explores the contribution of diverse-positioned methoxy substituents to the antimigratory and cytotoxic activities of this class. Synthesized analogues were tested in the MTT, cell cycle, and wound-healing assays. Previous studies on this class reported weak to medium antimitotic activity. Therefore, all compounds were subjected to tubulin polymerization assay and in silico molecular docking study at the colchicine binding site of tubulin. The 2-methoxy and 2,4-dimethoxy substitutions at the 2-arylpyrimido functionality enhanced the antimigratory activity in the 9-methoxy-substituted series like 6 and 9. The 3,4,5 trimethoxy substitutions at the 2-arylpyrimido group also significantly improved the antimigratory activity in the presence or absence of the 9-methoxy substitution as represented by 13 and 22, respectively. Docking experiments showed two distinct orientations at the colchicine binding site of tubulin. The first, achieved by 7 and 16–20, coincides with that of colchicine and positions the methoxy-substituted 2-aryl ring deep in the highly hydrophobic narrow end of the funnel-shaped binding pocket. On the other hand, 5, 6, 9–14 and 21, were oriented towards the wider opening of the binding pocket. Pyrimido[4,5 c]quinolin-1(2H)-ones are promising antimigratory hits with potential for future use to control metastatic breast cancers.