Antimicrobial resistance poses one of the most significant medical challenges for humanity. The current burden is overwhelming and is projected to escalate rapidly, with predictions for 2050 indicating 10 million deaths per year due to antibiotic-resistant microorganisms. Enhancing public awareness and education on this topic is crucial in efforts to mitigate this issue. In our study, we translated an existing questionnaire on antimicrobial resistance into Portuguese, validated it, and applied it between December 2020 and March 2021 to a group of Portuguese students (n = 112) and science teachers (n = 95). A majority of the students surveyed (65.1%) incorrectly believed that antibiotics could treat colds/flus. As anticipated, the teachers outperformed the students in the questionnaire. However, difficulties with this topic were evident in both groups. Most notably, the misconception that the human body becomes resistant to antibiotics was prevalent among most participants (77.0% of students and 68.4% of teachers). Consistent with previous studies in other populations and geographic locations, our research reveals a worrying lack of knowledge about antimicrobial resistance among Portuguese students and science teachers. Consequently, it is deemed urgent to implement effective measures to raise awareness and educate on this topic.