School-based tobacco prevention programs have been proven effective in reducing tobacco use. This evaluation aimed to assess the impact of an online tobacco prevention teacher training program on teachers and their students in Florida schools. A total of 344 teachers, including 72 K-3 grade teachers, 44 4th-5th grade teachers, and 228 6th-12th grade teachers completed the online training program and 323 (93.9%) were followed up to assess impacts on their 6,490 students. Results suggested that the online tobacco prevention education program for teachers was effective with high satisfaction and the impact on students was significant in improving knowledge and attitude about tobacco use and in increasing the proportion of 6–12th grade students who decided not to use tobacco. The evaluation study recommended the online education program be continued and expanded in the future.