Mycobacterium tuberculosis (Mtb) must sense and adapt to immune pressures such as acidic pH and reactive oxygen species (ROS) during pathogenesis. The goal of this study was to isolate compounds that inhibit acidic pH resistance, thus defining virulence pathways that are vulnerable to chemotherapy. Here we report that the acidic pH-dependent compound AC2P36 depletes intracellular thiol pools, sensitizes Mtb to killing by acidic pH, and potentiates the bactericidal activity of isoniazid, clofazimine, and oxidizing agents. We show that the pH-dependent activity of AC2P36 is associated with metabolic stress at acidic pH and a pH-dependent accumulation of intracellular ROS. Mechanism of action studies show that AC2P36 directly depletes Mtb thiol pools. These data support a model where chemical depletion of Mtb thiol pools at acidic pH enhances sensitivity to oxidative damage, resulting in bacterial killing and potentiation of antibiotics.