Exotoxins of Staphylococcus aureus belong to a family of bacterial proteins that act as superantigens by activating a large subset of the T-cell population, causing massive release of inflammatory cytokines. This cascade can ultimately result in toxic shock syndrome and death. Therapeutics targeting the early stage of the pathogenic process, when the superantigen binds to its receptor, could limit the severity of disease. We engineered picomolar binding affinity agents to neutralize the potent toxin staphylococcal enterotoxin B (SEB). A single immunoglobulin-like domain of the T-cell receptor (variable region, Vβ) was subjected to multiple rounds of directed evolution using yeast display. Soluble forms of the engineered Vβ proteins produced in Escherichia coli were effective inhibitors of SEB-mediated T-cell activation and completely neutralized the lethal activity of SEB in animal models. These Vβ proteins represent an easily produced potential treatment for diseases mediated by bacterial superantigens.