There is a relationship between teachers' competence in using Web 2.0 tools, the frequency of use of these tools in educational environments, and the increase in students' interest, attitude, and motivation towards the lesson in the context of educational technologies. Teachers need to use technological tools and equipment effectively in the learning environment, especially in making education more effective in science classes, enhancing student involvement, and establishing an interactive teaching environment. This research aimed to investigate the self-efficacy of biology teachers in using Web 2.0 tools according to different variables. The survey model, one of the quantitative research approaches, was used in the study. The study included 92 biology teachers from various schools around Turkey. Web 2.0 Tools Usage Competence Scale created by Çelik (2021) was used as the data collection tool. The independent t-test and variance analysis (ANOVA) were employed to analyze the data. The findings of this study show that there is no significant difference in biology teachers' Web 2.0 tools usage competence levels for their gender, the type of institution they work in, and the frequency of technology use. However, there are significant differences in biology teachers’ Web 2.0 tools usage competence levels for the duration of professional experience and exposure to training on Web 2.0 toolsBiology teachers with less professional experience scored higher on the scale compared to those with more experience. Furthermore, it was shown that teachers who got training on Web 2.0 technologies had greater competency ratings in utilizing Web 2.0 tools than teachers who did not get training.