Diverse inputs dictate macrophage transcriptional programming that impacts activation and relevant function, under homeostatic and disease conditions. My work addresses how stress-responsive transcription factor EB (TFEB) regulates macrophage gene expression following bacterial infection or exposure to cholinergic drugs. Using TFEB deficient macrophages, we show that TFEB is critical for mounting pro-inflammatory responses to Staphylococcus aureus infection. Mechanistically, the NADPH oxidase (PHOX)-dependent oxidative burst triggers TFEB activation, involving CD38 and NAADP-mediated Ca2+ release from intracellular stores. These findings reveal a previously unknown pathway, linking bacterial phagocytosis to macrophage phenotype and function via TFEB. In the absence of infection, we show that treatment of naïve macrophages with cholinergic agent PNU282987, an α7 nicotinic acetylcholine receptor (α7nACHR) agonist, induces pro-inflammatory gene expression, increasing Tnf, Ifnb1, Il1b and Il6 mRNA levels, without translating to increased cytokine secretion. However, primary macrophages and RAW264.7 cells showed no detectable α7nAChR expression, challenging the proposed α7nACHR -specific mode of action. We propose that PNU282987 may function through a non-receptor mediated pathway by activating stress responsive factor TFEB that involves ROS/H2O2 generation, implicating the MCOLN1-calcineurin pathway. These data offer critical insights into the pharmacological effects of PNU282987 beyond conventional receptor-based mechanisms. Overall, we describe novel mechanistic insights for macrophage TFEB activation as an integrator of distinct inputs, with important implications for inflammation and homeostasis.