The increasing prevalence of herbicide-resistant weeds has led to a search for new herbicides that target plant growth processes differing from those targeted by current herbicides. In recent years, some studies have explored the use of natural compounds from microorganisms as potential new herbicides. We previously demonstrated that tenuazonic acid (TeA) from the phytopathogenic fungus Stemphylium loti inhibits the plant plasma membrane (PM) H⁺-ATPase, representing a new target for herbicides. In this study, we further investigated the mechanism by which TeA inhibits PM H⁺-ATPase and the effect of the toxin on plant growth using Arabidopsis thaliana. We also studied the biochemical effects of TeA on the PM H⁺-ATPases from spinach (Spinacia oleracea) and A. thaliana (AHA2) by examining PM H⁺-ATPase activity under different conditions and in different mutants. Treatment with 200 μM TeA-induced cell necrosis in larger plants and treatment with 10 μM TeA almost completely inhibited cell elongation and root growth in seedlings. We show that the isoleucine backbone of TeA is essential for inhibiting the ATPase activity of the PM H⁺-ATPase. Additionally, this inhibition depends on the C-terminal domain of AHA2, and TeA binding to PM H⁺-ATPase requires the Regulatory Region I of the C-terminal domain in AHA2. TeA likely has a higher binding affinity toward PM H⁺-ATPase than the phytotoxin fusicoccin. Finally, our findings show that TeA retains the H⁺-ATPase in an inhibited state, suggesting that it could act as a lead compound for creating new herbicides targeting the PM H⁺-ATPase.