This paper explores the influence of Science Teaching Orientations (STOs) on Teacher Professional Knowledge (TPK) domains of five in-service Malawian secondary school science teachers. The study was grounded within Friedrichsten et al.’s (2011) definition of STOs and Gess-Newsome’s (2015) conceptualization of TPK. We gathered data using semi-structured interviews on the two dimensions of STOs: goals and purposes of science teaching, and beliefs about science teaching and learning. We used a questionnaire to gather data on the third dimension—Nature of Science (NoS). To understand the influence of STOs on TPK domains, we used data that we collected through classroom observations. The classroom observations were analysed inductively then deductively against the STOs dimensions and TPK domains. Results show that the first two dimensions of STOs influenced some TPK domains. There was no influence of the NOS on TPK domains. Although the teachers had correct views about the NOS, the lack of influence between the NOS and TPK domains raises questions about their Pedagogical Content Knowledge (PCK) for the NOS. We discuss the implications of the findings on teacher education and in-service science teacher professional development.