Bias-motivated aggression, particularly driven by sexual orientation, continues to have deleterious effects on society. Past research has identified perpetrator prejudice and victim nonconformity as risk factors for aggressive behavior. The current study sought to investigate effects of pepetrator sexual prejduice and gender conformity on aggression and social distancing toward women based on sexual orientation and gender expression. One hundred fifty-one undergraduate men and women participated in a competitive reaction-time task, during which they had an opportunity to shock an ostensible opponent as a measure of aggression. Participants were assigned to one of four opponent conditions (masculine, lesbian; feminine, lesbian; masculine, straight; feminine straight). Analyses revealed high prejudice women aggressed more toward heterosexual than lesbian opponents and that low gender conforming men aggressed more toward feminine than masculine opponents. Additionally, findings emerged regarding mens distancing behavior toward women. Findings are discussed in terms of variables that encourage or inhibit aggressive responding.