NADPH oxidase 2 (NOX2) is a multi-subunit enzyme composed of a catalytic flavocytochrome core and regulatory subunits that plays a pivotal role in phagocytes where it is responsible for the production of reactive oxygen species (ROS). Upon cellular stimulation, NOX2 components assemble at the cell membrane, enabling electron transfer from cytoplasmic NADPH to oxygen, and resulting in the generation of superoxide anions that are toxic to cells. This process plays an important role in the elimination of damaging microorganisms associated with bacterial infection. This activity of NOX2 is tightly regulated by phosphorylation events that are mediated by various kinases, including phosphorylation of its subunits p47phox and p67phox. Phosphorylation drives translocation of these subunits and their assembly at the cell membrane. Peroxiredoxin 6 (Prdx6), a thiol-dependent peroxidase that is selenium- and heme-free, also exhibits both calcium-independent acidic phospholipase A2 (aiPLA2) and lysophosphatidylcholine acyltransferase (LPCAT) activities that contribute to cellular phospholipid metabolism. The role of the Prdx6-aiPLA2 activity in NOX2 activation has been demonstrated through the use of both the PLA2 inhibitor MJ33 and Prdx6 “knockout” mouse models. aiPLA2 activity of Prdx6 liberates free fatty acids, including arachidonic acid (AA), and lysophospholipids. Unsaturated fatty acids like AA induce conformational changes in p47phox, enabling its interaction with p22phox and facilitating the binding of the rac-GTP-p67phox complex to the NOX2-binding site. These free fatty acid and lysolipid products generated from aiPLA2 activity can modulate NOX2 activation via distinct mechanisms, thereby highlighting a critical interplay between regulation of NOX2 and Prdx6 aiPLA2 activities.