Black root rot, caused by Thielaviopsis basicola, is a serious disease of cotton in Australia, causing necrosis of the root cortex, delayed seedling development and fruit set, and yield loss when severe. We investigated the potential for acibenzolar-S-methyl to induce resistance in cotton against T. basicola in naturally infested soil. In pot experiments, soaking seeds in solutions of acibenzolar-S-methyl (25 or 50 µg/mL for 3 to 5 h before planting) consistently reduced disease severity on tap roots by 20–30%. In field experiments, acibenzolar-S-methyl was applied either as a spray over the seed during sowing (in-furrow spray), as a seed dressing, by the soaking method, or as foliar sprays over seedlings. In the field, seed-soaking reduced the severity of black root rot on tap roots by 33%. The in-furrow spray (25 µg acibenzolar-S-methyl/mL at 550 L/ha) reduced the severity of symptoms on tap roots by 24%, increased the number of relatively healthy lateral roots by 350% and increased fruit number by 29%. Seed dressing with 3.3 mg acibenzolar-S-methyl/kg seed did not affect disease severity, whereas 6 mg/kg reduced the severity on tap roots by 20%. The foliar sprays were ineffective. To our knowledge, this is the first report of the induction of systemic acquired resistance against a soilborne plant pathogen in the field using a chemical seed treatment. With appropriate rates and application methods, acibenzolar-S-methyl has potential as a component in an integrated disease management strategy for black root rot of cotton.