In Spain, students pursuing a career in athletic training, physical education, or scientific evaluation of sports enroll in a bachelor’s degree in sports sciences. This degree provides knowledge and skills in a broad array of sports settings and promotes research-based interdisciplinary knowledge. However, the student’s syllabus rarely includes specific academic training on anti-doping regulations or doping prevention. The purpose of this study was to assess the anti-doping knowledge of the students undertaking a bachelor’s degree in sports sciences in Spanish universities. One thousand two hundred and thirty-three bachelor students in sport science (907 males, 322 females, and 4 participants with non-binary sex) from 26 Spanish universities completed a validated questionnaire about general anti-doping knowledge. The questionnaire is an adapted version of the Play True Quiz of the World Anti-Doping Agency and contains 37 multiple-choice questions. The score obtained in the questionnaire was transformed into a 0–100-point scale. The questionnaire was distributed among students within each university by a faculty member and it was filled out online. Students obtained a score of 65.8 ± 10.10 points (range = 32–92 points). There was an effect of the course in the score obtained (p < 0.001). Students of the first course (63.6 ± 9.5 points) had lower scores than the remaining courses (p < 0.037) while the students of the fourth course obtained the highest scores (68.7 ± 9.5 points; p < 0.019). The students with an itinerary on sports performance were the respondents with the highest anti-doping knowledge (67.2 ± 10.2) points, followed by the students with an itinerary on health (66.7 ± 9.5 points). The knowledge of basic anti-doping rules and doping prevention strategies of the bachelor students in sports sciences in Spain was suboptimal. Increasing doping prevention information in the syllabus of the bachelor’s degree in sports sciences is essential as these future professionals will directly work with populations at risk of doping.