Lime is one of the abiotic stress factors limiting the cultivation of many fruit species. High lime content causes significant yield losses since plants are unable to effectively perform photosynthesis. This study aimed to determine the effect of calcium carbonate (CaCO3) applications at different levels in blackberry in vitro conditions. Plants were grown at three different CaCO3 levels: as 1 gl−1, 3 gl−1, and 5 gl−1 in MS rooting medium. The plants of ‘Chester’ blackberry were micropropagated by tissue culture techniques referred to as “nodal culture”. Plant height, plant diameter, plant fresh and dry weight, root number, root length, leaf relative water content (LRWC), chlorophyll content, hydrogen peroxide (H2O2), and malonaldehyde (MDA) concentrations were determined in plants. According to the results, the highest values in morphological parameters such as plant height (3.96 cm), fresh plant weight (0.845 g), dry plant weight (0.157 g), and root length (15.50 cm) were obtained in control plants. The highest H2O2 (6.957 µmolg−1 FW) and MDA (0.493 nmolmlg−1) concentrations were obtained from 5 gl−1 CaCO3 + 0.5 gl−1 KHCO3 application. In conclusion, the increasing CaCO3 levels dramatically decreased plant growth and development in MS conditions. According to our results, it has been determined that high CaCO3 levels negatively affect plant growth. It was determined that 1 gl−1 CaCO3 + 0.5 gl−1 KHCO3 application showed similar growth compared to our control plants. Based on these results, it can be said that the ‘Chester’ blackberry variety has low tolerance to soils with high lime content.