Biofilm growth is an emergent property and frequently unpredictable, particularly in multi-species communities. Pseudomonas aeruginosa, Pseudomonas fluorescens, and Escherichia coli are well studied in monoculture yet co-exist in polymicrobial biofilms in clinical and environmental settings, including catheters, the airways of cystic-fibrosis patients, and wastewater systems. Studying these complex environments requires imaging approaches capable of resolving both cellular detail and large-scale organisation. In this work, we employed cross-scale and multi-modal imaging strategies to examine the internal architecture of bacterial biofilms. By combining laser-scanning and spinning-disk confocal microscopy, mesoscopy with the Mesolens and light-sheet fluorescence imaging, we have gone beyond previous studies to visualise biofilm structures across a range of spatial scales. The inclusion of spinning-disk and light-sheet modalities enabled enhanced depth, speed and more sensitive imaging than conventional approaches, improving our ability to resolve intricate biofilm structures and cell-cell dynamics in phylogenetically distinct bacteria. Together, these methods bridge the gap between whole-biofilm imaging and cellular-level detail, capturing global organisation alongside internal architecture. Integrating these imaging techniques revealed nutrient transport channels and spatial variation within polymicrobial biofilms that were not easily observed using a single method. Comparative imaging of mono- and dual-species biofilms also showed interactions between species influence internal organisation and display species-dependent colonisation patterns, with P. fluorescens occupying channel regions of E. coli, while P. aeruginosa and E. coli remained spatially separate. Current work focuses on understanding the effect of nutrient, chemical and environmental stressors within these structures and expanding beyond laboratory strains to a panel of pathogenic clinical isolates.