This record is Part 1 of the data associated with the publication:Janež, Škrlj, Osojnik et al. MicroICS: Extracting predictive information from structural features of Listeria monocytogenes biofilms for strain identification and biological context associations. This part provides the data supporting image acquisition, metadata, image preparation, and structural feature extraction. Feature files are provided for both MicroICS, the tool developed in this study, and BiofilmQ, which is included as a comparison algorithm to demonstrate that the classification approach is not tool-specific and is suitable for features calculated by either method. lsm_to_tif.ijm - ImageJ macro script to export images from multi-image LSM files to single-image TIFF format. 17_11_2023_3D_z_21 - Directory containing all 3D microscopy images of biofilms used for classifier comparison. 17_11_2023_3D_z_21_metadata.xlsx - Metadata associated with the image dataset 17_11_2023_3D_z_21, including experimental conditions and strain information. datafile_MicroICS_features_benchmark_and_stat_analy.tsv - Structural features calculated by MicroICS for the 17_11_2023_3D_z_21 dataset, used for classifier comparison and statistical analysis. datafile_MicroICS_BiofilmQ_features.tsv - Structural features calculated by both MicroICS and BiofilmQ for the 17_11_2023_3D_z_21 dataset, provided for algorithm comparison. datafile_BiofilmQ_features.tsv - Structural features calculated exclusively by BiofilmQ for the 17_11_2023_3D_z_21 dataset.