The purpose of this study is to enhance online biology learning with mobile augmented reality (AR) applications and to assess the impact of mobile AR applications on students’ motivation, self-efficacy, and attitudes toward biology learning. Students were interviewed, and the usefulness of mobile AR applications was evaluated using a quasi-experimental pretest–posttest approach. The study group consists of 71 high school students, 26 in the control group and 45 in the experimental group, attending a public high school in the Western Black Sea Region of Turkey during the academic year 2020–2021. The self-efficacy ratings of the experimental group of students who participated in mobile AR-based biology learning were statistically higher than those of the control group after a 12-week trial. However, there were no statistically significant differences between experimental and control group students’ motivation and attitudes toward biology learning. In addition, as a result of student interviews, mobile AR applications were deemed innovative, non-distracting, successful in knowledge acquisition, engaging, intriguing, and entertaining, boosting information retention, concretizing the subject, and facilitating learning.