The purpose of this study was to assess the level of knowledge about prevention of mother-to-child transmission option B+ and associated factors among antenatal care clients in Kombolcha town; 2017. Methods: An institutional-based cross-sectional study design was conducted from May 1 to 30, 2017. Simple random sampling techniques were used to select 129 study subjects from May 1 to 30, 2017. A chi-square test was done by using SPSS 20 to assess the association between the dependent and independent variables. Frequency tables, pie chart, and graphs were used to present the findings of the study. Results: Among the total study participants, 61% had adequate knowledge about prevention of mother-to-child transmission Option B+. This study found a strong positive association between knowledge of prevention of mother-to-child transmission B+ option and age, educational status, occupational status, number of ANC follow-up visits for the current pregnancy and number of parity. Conclusion: This study showed more than half of these study participants had adequate knowledge about the prevention of mother-to-child transmission B+ option. Age, educational status, employment, parity and number of antenatal care visits had a statistically significant association with pregnant womens' knowledge of prevention of mother-to-child transmission B+ option. Therefore, it is better to design to give health education on prevention of mother-to-child transmission B+ option particularly for older age, uneducated, unemployed, partiy of one pregnant women.