Background: Trachoma is the leading infectious cause of blindness worldwide, particularly impacting the poorest nations, including Ethiopia. Objective: To assess knowledge, practice, and associated factors of trachoma transmission and prevention among primary school students in Addis Zemen town, South Gondar, Northwest Ethiopia, 2022. Methods: An institution-based cross-sectional study was conducted on 407 randomly selected primary school students (grades 5-8), aged 10 to 24 years. Data collection included face-to-face interviews using structured questionnaires and an observation checklist. Epi-data version 4.2.0.0 and SPSS version 20 were used to enter and analyze the collected data, respectively. The data were analyzed using the descriptive statistical method and using bivariable and multivariable logistic regression models. Variables with a P-value <.05 with a 95% CI were considered to have statistical significance. Result: The study has a response rate of 98.25%, with a total of 400 respondents. 74.00% of the respondents knew about trachoma transmission and prevention, and 71.50% practiced preventive measures, such as face washing. Respondents in Grades 7 and 8, aged 12 to 24 years (AOR: 2.67, 95% CI: 1.93, 4.64), information about trachoma (AOR: 2.30, 95% CI: 1.56, 4.21), and urban residence (AOR: 3.42, 95% CI: 2.56, 5.23) were determinants of knowledge regarding trachoma transmission and prevention. Meanwhile, the mother’s occupation (government employee) (AOR: 2.50, 95% CI: 1.83, 6.91) and overall knowledge about trachoma (AOR: 4.87, 95% CI: 2.95, 8.53) were significant predictors of the practice of trachoma transmission prevention. Conclusion: While the level of knowledge and practice regarding trachoma transmission and prevention was relatively high, 26.00% of respondents still lacked adequate knowledge, and 28.50% did not practice prevention measures. To reduce the spread of trachoma and enhance public health outcomes, targeted interventions focusing on education about transmission and prevention strategies such as promoting face washing and proper sanitation should be prioritized in this area.