English vocabulary development plays a vital part in language learning in many contexts, which seems to be difficult for learners who study English as a second or foreign language (English as a foreign language; EFL). Vocabulary development also includes knowledge of word-formation. Consequently, understanding current prefix knowledge of second language learners may be beneficial for scholars who are finding a tool for the learners to learn new words. This study aims to investigate the current knowledge of English prefixes focusing on “in- and im-” and the ability of EFL students to distinguish the meaning between them. The participants of this study were 65 third-year students who are studying in the English major at Naresuan University. The researcher used tests adapted from Cook (2016) as an instrument to find out the students’ prefix content knowledge. The results of this study demonstrated that the 3^rd year students majoring in English could apply the prefix knowledge that they have learned in the morphology course in doing the tests. Besides, the findings also suggested that students could distinguish the meaning of the same prefixes with different meanings. Finally, the study concluded that teaching prefixes both implicitly and explicitly in the classroom may help improve learners’ vocabulary acquisition. However, teachers or course developers may need to develop additional teaching materials that also promote students’ affix knowledge.