The pedagogical content knowledge (PCK) of teachers influences students’ achievement of the learning outcomes. This study examined teachers’ perceived enactment of PCK in biology. The quantitative survey design was adopted by using a Likert-scale questionnaire consisting of six components of PCK, namely curricular saliency, students’ prior knowledge, what makes the subject easy or difficult, representations, conceptual teaching strategies, and assessment. Data on teachers’ perceived enacted PCK (ePCK) were collected from 54 biology teachers selected from 14 secondary schools in three districts of Lusaka province. The data were analyzed by computing descriptive and inferential statistics using Statistical Package for the Social Sciences (SPSS) software. The findings revealed that: (a) respondents’ perceived ePCK was high (M = 4.29, SD = .37), (b) respondents’ perceived ePCK was not influenced by gender, teaching experience, and type of school, (c) respondents’ perceived ePCK was influenced by their academic qualifications, (d) the component students’ prior knowledge and misconceptions was the most enacted component, and (e) the component what makes the subject easy or difficult was the least enacted component. The results highlight areas in teachers’ PCK that require enhancement. The study recommends using teacher professional development to enhance teachers’ ePCK in the component what makes the subject easy or difficult to understand. Further research may use larger samples and more data sources to increase the validity of the findings.