This dataset contains the raw and processed experimental data, along with the analysis code, for the publication titled “A Method for Optical Quantification of Local Oxygen Limitations in Multiphase Bioreactors.” The data were obtained from optical measurements based on the reversible redox reaction of methylene blue in gas–liquid stirred tank bioreactors. They include time-resolved colourisation data used to derive spatial information on oxygen supply heterogeneities under varying hydrodynamic conditions (e.g. different impeller speeds). The dataset is structured into raw measurement files, processed results, and scripts for data evaluation and visualisation, enabling reproducibility and further analysis.