Preschool vision screening is carried out by optometrist in Malaysia. However, inadequate manpower limits the ability of this screening to be conducted nationwide. Teachers’ involvement in the screening team could be beneficial to overcome this issue. Therefore, it is important to understand teachers’ knowledge, attitude, and practice (KAP) on vision screening. This study aimed to assess preschool teachers’ level of KAP and identify the contributing factors. A cross-sectional study was conducted among public and private preschool teachers in Malaysia. A self-administered online questionnaire was used as the data collection tool. The contributing factors affecting the KAP level were determined using binary logistic regression analysis. The majority of the preschool teachers were in the high-level category for knowledge (74.1%), attitude (75.0%), and practice (81.2%). Factors contributing to the level of knowledge were teachers from private preschools and serving at preschools conducting vision screening. Being female, teachers with a non-education field of study and without a history of eye problems/diseases were associated with attitude level. Meanwhile, factors contributing to the practice level were age, Malay teachers, involvement in vision screening training, and serving at preschools conducting vision screening. Training and exposure to vision screening can increase teachers’ KAP levels regarding vision screening.