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The isoflavone biochanin A was previously shown to promote weight gain in growing steers by selectively inhibiting rumen bacteria-like growth-promoting feed antibiotics. The hypothesis that biochanin A inhibited the action of drug efflux pumps was tested by enumerating tetracycline-insensitive bacteria from steers in a subacute rumen acidosis (SARA) challenge. Steers (<i>n</i> = 3/group) treatment groups were forage only, SARA control, SARA with monensin (0.2 g d⁻¹), and SARA with biochanin A (6.0 g d⁻¹). As the steers were stepped up from the forage-only basal diet to 70% cracked corn, the number of rumen bacteria enumerated on two tetracycline-containing media types (nutrient glucose agar and tetracycline, and bile esculin azide and tetracycline) increased (<i>p</i> < 0.05) from as little as 1.7(10⁵) to as great as 6.7(10⁶) cfu mL⁻¹ on the nutrient glucose agar in the SARA and monensin control groups. The biochanin A group maintained the same number of tetracycline-insensitive bacteria as the forage-only controls (<i>p</i> > 0.05). The effects were similar to the more selective media type, but the differences were smaller. These results support the hypothesis that biochanin A inhibits the activity of drug efflux pumps in vivo.