Leukotrienes (LTs) and prostaglandin (PG)E2, produced by 5-lipoxygenase (5-LO) and microsomal prostaglandin E2 synthase-1 (mPGES-1), respectively, are key players in inflammation, and pharmacological suppression of these lipid mediators (LM) represents a strategy to intervene with inflammatory disorders. Previous studies revealed that the benzenesulfonamide scaffold displays efficient 5-LO-inhibitory properties. Here, we structurally optimized benzenesulfonamides which led to an N-phenylbenzenesulfonamide derivative (compound 47) with potent inhibitory activities (IC50 = 2.3 and 0.4 μM for isolated 5-LO and 5-LO in intact cells, respectively). Compound 47 prevented the interaction of 5-LO with its activating protein (FLAP) at the nuclear envelope in transfected HEK293 cells as shown by in situ proximity ligation assay. Comprehensive assessment of the LM profile produced by human macrophages revealed the ability of 47 to selectively down-regulate pro-inflammatory LMs (i.e. LTs and PGE2) in M1 but to enhance the formation of pro-resolving LMs (i.e. resolvins and maresins) in M2 macrophages. Moreover, 47 strongly inhibited LT formation and cell infiltration in two in vivo models of acute inflammation (i.e., peritonitis and air pouch sterile inflammation in mice). Together, 47 represents a novel LT biosynthesis inhibitor with an attractive pharmacological profile as anti-inflammatory drug that also promotes the biosynthesis of pro-resolving LM.