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This study examined the effect of Cooperative learning (CL) on content knowledge and teaching self-efficacy of EFL pre-service teachers using a pre-test/post-test quasi-experimental design. The experimental group (N = 35) and the control group (N = 30) were randomly selected from two Cambodian regional teacher training centres. For 16 weeks, the experimental group was exposed to CL while the control group participated in lecture-based learning. Data was collected before and after the experiment through an achievement test and an adapted scale on teaching self-efficacy. The ANCOVA results revealed that the EFL pre-service teachers in the experimental group outperformed their conventionally trained counterparts in terms of grammar and vocabulary achievement and teaching self-efficacy. This study adds to the existing literature, showing that CL significantly contributes to the increase in content knowledge and teaching self-efficacy among EFL pre-service teachers and highlights the need for applying CL in pre-service instruction.