Mitochondrial biogenesis initiates within hours of T cell receptor (TCR) engagement and is critical for T cell activation, function, and survival; yet, how metabolic programs support mitochondrial biogenesis during TCR signaling is not fully understood. Here, we performed a multiplexed metabolic chemical screen in CD4+ T lymphocytes to identify modulators of metabolism that impact mitochondrial mass during early T cell activation. Treatment of T cells with pyrvinium pamoate early during their activation blocks an increase in mitochondrial mass and results in reduced proliferation, skewed CD4+ T cell differentiation, and reduced cytokine production. Furthermore, administration of pyrvinium pamoate at the time of induction of experimental autoimmune encephalomyelitis, an experimental model of multiple sclerosis in mice, prevented the onset of clinical disease. Thus, modulation of mitochondrial biogenesis may provide a therapeutic strategy for modulating T cell immune responses. Display omitted •High-throughput chemical screen discovers modulators of mitochondrial mass•PP blocks mitochondrial mass increase during T cell activation•PP inhibits T cell function by targeting pyruvate oxidation•PP impairs helper T cell differentiation, preventing the onset of EAE Kurmi et al. present a high-throughput platform quantifying mitochondrial mass in early T cell activation. Pyrvinium pamoate (PP) emerges as a potent inhibitor of mitochondria, disrupting pyruvate oxidation and impairing T cell function. PP impacts helper T cell differentiation to prevent EAE onset, revealing modulation of mitochondria as a potential target for autoimmunity.