The experience of graduate degree lecturers in the natural sciences when they switched to online teaching during the Covid-19 pandemic is described. The shift to online teaching throughout the pandemic provided an opportunity to evaluate how lecturers integrate technology into their teaching and what they need to improve their remote teaching. This study used a twofold perspective of TPACK (Technological Pedagogical Content Knowledge) and self-efficacy in online education. Its data were derived from pre-and post-questionnaires, comprising closed and open-ended questions, given at the start and end of the semester. We found that lecturers focused on learning and applying technological and techno-pedagogical knowledge but paid less attention to the integration of three components: technology, pedagogy, and scientific content. Although no statistically significant differences in lecturers’ perceived self-efficacy was found between the start and the end of the semester, at the end of the semester we found a statistically significant correlation between the variables involved in building self-efficacy in online teaching: (1) satisfaction with online teaching and the belief that (2) technology promotes teaching, student interactions, participation, and engagement. Our results enabled us to identify the knowledge aspects that lecturers implemented initiatively and to better understand what aspects required more professional development training. In addition, the results emphasized the importance of developing the lecturers’ self-efficacy for online teaching. These insights can help to improve and enhance online teaching in higher education.