The NADPH oxidase enzyme complex, NOX2, is responsible for reactive oxygen species production in neutrophils and has been recognized as a key mediator of inflammation. Here, we have performed rational design and in silico screen to identify a small molecule inhibitor, Phox-I1, targeting the interactive site of p67 phox with Rac GTPase, which is a necessary step of the signaling leading to NOX2 activation. Phox-I1 binds to p67 phox with a submicromolar affinity and abrogates Rac1 binding and is effective in inhibiting NOX2-mediated superoxide production dose-dependently in human and murine neutrophils without detectable toxicity. Medicinal chemistry characterizations have yielded promising analogs and initial information of the structure-activity relationship of Phox-I1. Our studies suggest the potential utility of Phox-I class inhibitors in NOX2 oxidase inhibition and present an application of rational targeting of a small GTPase-effector interface.