Chlamydia trachomatis (Ct) is a bacterial intracellular pathogen responsible for a plethora of diseases ranging from blindness to pelvic inflammatory diseases and cervical cancer. Although this disease is effectively treated with antibiotics, concerns for development of resistance prompt the need for new low-cost treatments. Here we report the activity of spilanthol (SPL), a natural compound with demonstrated anti-inflammatory properties, against Ct infections. Using chemical probes selective for imaging mitochondrial protein sulfenylation and complementary assays, we identify an increase in mitochondrial oxidative state by SPL as the underlying mechanism leading to disruption of host cell F-actin cytoskeletal organization and inhibition of chlamydial infection. The peroxidation product of SPL (SPL endoperoxide, SPL^E), envisioned to be the active compound in the cellular milieu, was chemically synthesized and showed more potent anti-chlamydial activity. Comparison of SPL and SPL^E reactivity with mammalian peroxiredoxins, demonstrated preferred reactivity of SPL^E with Prx3, and virtual lack of SPL reaction with any of the reduced Prx isoforms investigated. Cumulatively, these findings support the function of SPL as a pro-drug, which is converted to SPL^E in the cellular milieu leading to inhibition of Prx3, increased mitochondrial oxidation and disruption of F-actin network, and inhibition of Ct infection.