Modulation of the cyclooxygenase (COX) pathway, particularly the regulation of COX-2 activity and expression, is central to anti-inflammatory therapy. In this study, the COX-2 inhibitory potential and COX-2/COX-1 selectivity of a panel of 43 structurally related 2-styrylchromones (2-SC), a family of oxygen-containing heterocyclic compounds and vinylogues of flavones (2-arylchromones) were evaluated. COX-2 and COX-1 activities were assessed using in vitro enzymatic and ex vivo human whole blood assays, complemented by the analysis of COX-2 expression in lipopolysaccharide (LPS)-stimulated human leukocytes. Structure-activity relationships were established whenever possible. In the enzymatic assay, 2-SC B12 and B13 were the most active compounds, with IC50 values ≤ 1 μM. These results suggest that the presence of catechol groups at C-7 and C-8 on the A-ring, as well as at C-3' and C-4' on the B-ring, plays a key role in the inhibition of COX-2 enzymatic activity. Both compounds were selective for COX-2, though B12 showed the highest selectivity index. In the complex matrix of human whole blood, B1 was the only 2-SC that consistently displayed inhibitory activity in both enzymatic and whole blood assays. At the cellular level, several 2-SC significantly downregulated LPS-induced COX-2 expression in human leukocytes. Notably, 2-SC B6, B7 and B9 inhibited both COX-2 activity and expression, and also reduced prostaglandin E2 production, suggesting a dual modulatory effect on this inflammatory pathway. Overall, these findings identify the 2-SC scaffold as a promising and underexplored chemical platform for the rational design of selective COX-2 modulators and innovative anti-inflammatory agents.