In the course of digitalization, new technologies and innovations are continuously introduced to the educational sector. For instance, augmented reality (AR) is increasingly applied in science teaching in both school and higher education. Combining real and virtual content potentially enhances interactivity and understanding of the learning process. This teaching and learning approach can positively impact various learning outcomes, such as learning gains and motivation. This paper aims to investigate the positive learning effects of AR using a game-based AR learning environment: “Beat the Beast”. In line with the concept of an education for sustainable development (ESD) topic, microplastics, this learning environment follows an interdisciplinary approach, combining the subjects of biology, chemistry, and engineering. To determine and distinguish the effects of implementing the technology AR in science learning environments as well as the principles of game-based learning, we contrasted the learning environment into two factors: one with AR and one without AR and the other with a game and without a game. A quasi-experimental design with 203 pre-service teachers of the first semester of all subjects was chosen to evaluate the four different types of settings with questionnaires on motivation, technology acceptance, user engagement, cognitive load, computer self-efficacy, knowledge, and ESD outcomes in higher education. Our research demonstrates that although augmented reality (AR) imposes a relatively elevated cognitive load, it does not negatively affect learning effects. In spite of the increased cognitive load, learners in AR settings do not exhibit lesser knowledge acquisition compared to those in alternative environments. Moreover, our investigation highlights AR’s potential to amplify motivation and user engagement. Contrary to expectations, in the context of the selected subject matter and target audience, game-based adaptations of the educational environment fail to enhance learning outcomes. These versions actually underperform compared to other formats in both motivational and engagement metrics.