Background: Hookah smoking is an emerging threat to the health of college students in the United States. Nonetheless, college students are not aware of the harm of using hookah, and it is likely that users use other substances concurrently. Limited research examined the associations between other substance use, substance use prevention education, and hookah smoking among this population. Thus, the purpose of the present study is to examine the extent of recent hookah use and the relationships between recent hookah use and both other substance use and substance use prevention education among college students. Methods: Participants were 765 college students at a Midwestern, metropolitan university and were recruited by the university wellness center. Students completed the American College Health Association National College Health Assessment II [ACHA-NCHA], a 65-item survey including health behaviors and demographics, in classrooms. Logistic regression analyses were performed to examine study research questions. Results: Results indicated that 14.2% of participants reported using hookah in the past 30 days. Recent hookah smoking was related to substance use including cigarettes, cigars, smokeless tobacco products, alcohol, and marijuana. The final logistic regression model indicated that recent hookah use was associated with recent cigarette use, recent alcohol use, and recent cigar use. Additionally, participants who had received tobacco prevention education were 1.6 (95% CI = 1.015-2.535) times more likely to report recent hookah smoking after controlling for demographics and recent substance use. Conclusions: Interventions on substance use prevention that include comorbid substance use may be warranted on college campuses. Tobacco prevention education programs should include or emphasize harm of hookah smoking and evaluate the effects on hookah smoking behaviors.