The aim of this experimental study was to compare the attitudes of prospective Science teachers towards Chemistry Laboratory and Chemistry Lesson delivered via inquiry-based instruction method in real and virtual laboratories. The study was conducted in 2013-2014 academic year and adopted Mixed Methods design based on both quantitative and qualitative data. The participants of the study comprised 34 freshman student teachers of Science at a faculty of Education in a state university in Turkey. The purposefully selected participants were assigned as Group-1 (N=17) and Group-2 (N=17). While the Group-1 was instructed via inquiry-based instruction in a real laboratory, the Group-2 was taught utilizing the same instruction method in a virtual laboratory. In the study, 8 different experiments were conducted in the both real and virtual laboratories using some related software. As the data collection tools, the Attitude Towards Chemistry Lesson Scale; the Attitude Towards Chemistry Laboratory Scale, and a semi-structured interview form were used. The results revealed that although all the prospective Science Teachers' (PST) attitudes toward Chemistry Lesson and Chemistry Laboratory improved significantly in both real and virtual laboratory environments, the improvement in their scores in a real laboratory environment was significantly higher than that in the virtual laboratory. The semi-structured interview results were coherent with the results of the scales.