Background: Little research has focused specifically on alcohol, tobacco, and other drug (ATOD) use of lesbian and bisexual women in general or the college subgroup specifically. Previous research shows sexual minority women at increased risk when compared to their heterosexual counterparts. Objectives: The purpose of the current study was to compare ATOD use of lesbian, bisexual, and heterosexual undergraduate college women. Methods: A secondary analysis of three fall semesters (2009–2011) of American College Health Association-National College Health Assessment (ACHA-NCHA-II) data was conducted. Women who self-identified as gay/lesbian (N = 538), bisexual (N = 1579), and heterosexual (N = 40,869) were compared on ATOD use. Chi-square Tests were used to analyze differences between the groups and logistic regression determined odds ratios of ATOD use. Results: Bisexual women had greater odds of using alcohol, tobacco, and marijuana than heterosexual women and lesbians. They also had greater odds of using all illicit drugs (except steroids) and misusing prescription drugs than heterosexual women and greater odds of using amphetamines other than meth, sedatives, and ecstasy when compared to lesbians. Lesbians had greater odds of using tobacco, marijuana, sedatives, hallucinogens, other illegal drugs and misusing prescription drugs than heterosexual women. Conclusions/Importance: The study confirms increased ATOD use among sexual minority women as compared to their heterosexual counterparts with bisexual women having the highest use. This is the first study to examine misuse of prescription drugs among a large number of lesbian and bisexual college women, and contributes to the scant literature addressing college women's ATOD use by sexual orientation.