The current study utilized a pre-and post-intervention design to assess pre-service teachers' self-efficacy, perceived content knowledge, and perceived pedagogical knowledge before and after a teaching practicum in the Pakistani context. The data were collected in two phases. In the pre-practicum phase, data were collected from 419 pre-service teachers enrolled in B. Ed, M. Ed, and MA Education programs to assess the general level of self-efficacy for teaching ESD-related concepts, their perceived content knowledge, and perceived pedagogical knowledge concerning ESD. In the post-practicum phase, the data were collected from 215 participants who participated in the first phase and consented to participate in the second phase. One-way ANOVA, Independent sample t-test and paired sample t-test were utilized for data analysis. Results suggest significant differences in pre-service teachers' self-efficacy based on the university, program, and age. Similarly, a difference was found between pre-service teachers' perceived content knowledge based on the university they were enrolled in. The difference in pedagogical knowledge was also found based on university and gender. Further, the post-practicum analysis suggests a significant difference in pre-service teachers' self-efficacy, perceived content knowledge, and pedagogical knowledge after undergoing a teaching practicum. Conclusion, limitations, and recommendations are provided at the end of the paper.