Background Effective implementation strategies are needed to enhance the success of evidence-based prevention programs. The current study evaluates the effects of two implementation strategies on teachers' implementation of an evidenced-based HIV intervention. Methods Using our 7-item pre-implementation school screening tool, we identified teachers who were at-risk for not implementing the Focus on Youth HIV-risk reduction intervention curriculum which targets grade six through grade 8 students. After completing a two-day curriculum workshop, 81 low- and moderate-performing teachers were randomly assigned to one of four experimental conditions and were asked to teach the two-month intervention curriculum. This optimization trial examines the impact of two implementation strategies: biweekly monitoring/feedbacks (BMF) and site-based assistance/mentorship (SAM). The primary outcome is implementation fidelity defined as number of core activities taught. Linear mixed-effects model was used to examine the association of the implementation strategies with implementation fidelity. Results BMF and SAM were significantly associated with teachers' implementation fidelity. Teachers who received both BFM and SAM taught the greatest numbers of core activities (15 core activities on average), followed by teachers who received either BMF (6.9 activities) or SAM (7.9 activities). Teachers who did not receive BMF or SAM taught the lowest numbers (4.1 activities). Teachers' sustained implementation of FOYC in the prior school year was related to increased implementation fidelity during the optimization trial. Teachers' confidence in implementing five core activities, attitudes toward sex education in schools, and perceived principal support were significantly related to increased self-efficacy, which in turn was related to teachers' fidelity of implementation before the optimization trial. Conclusion BMF and SAM are effective in promoting teachers' implementation of youth evidence-based interventions. Researchers and future program implementers should consider teacher training, teachers' attitudes toward sex education, perceived principal support, and self-efficacy when attempting to maintain the effects of teacher-delivered interventions in schools.