Triclosan (TCS) is an antimicrobial used widely in hospitals and personal care products, at ~10 mM. Human skin efficiently absorbs TCS. Mast cells are ubiquitous key players both in physiological processes and in disease, including asthma, cancer, and autism. We previously showed that non-cytotoxic levels of TCS inhibit degranulation, the release of histamine and other mediators, from rat mast cells (RBL-2H3), and in this study we replicate this finding in human (HMC-1.2) mast cells. Our investigation into the molecular mechanisms underlying this effect led to the discovery that TCS disrupts ATP production in RBL-2H3 cells in glucose-free, galactose-containing media (95% CI EC50 = 7.5-9.7 μM), without causing cytotoxicity. Using these same glucose-free conditions, 15 μM TCS dampens RBL-2H3 degranulation by 40%. The same ATP disruption was found with human HMC-1.2 cells (EC50 4.2-13.7 μM), NIH-3T3 mouse fibroblasts (EC50 4.8-7.4 μM), and primary human keratinocytes (EC50 3.0-4.1 μM) all with no cytotoxicity. TCS increases oxygen consumption rate in RBL-2H3 cells. Known mitochondrial uncouplers (e.g., CCCP) previously were found to inhibit mast cell function. TCS-methyl, which has a methyl group in place of TCS’s ionizable proton, affects neither degranulation nor ATP production at non-cytotoxic doses. Thus, triclosan’s effects on mast cell function are due to its proton ionophore structure. Also, 5 μM TCS inhibits thapsigargin-stimulated degranulation of RBL-2H3 cells: further evidence that TCS disrupts mast cell signaling. Our data indicate that TCS is a mitochondrial uncoupler, and TCS may affect numerous cell types and functions via this mechanism.