The functional properties of biofilms are intimately related to their spatial architecture. Structural data are therefore of prime importance to dissect the complex social and survival strategies of biofilms and ultimately to improve their control. Confocal laser microscopy (CLM) is the most widespread microscopic tool to decipher biofilm structure, enabling noninvasive 3D investigation of their dynamics down to single cell scale. The emergence of fully automated high content screening (HCS) systems, associated with large-scale image analysis, radically amplifies the flow of available biofilm structural data. In this contribution, we present an HCS-CLM protocol used to analyze biofilm 4D structural dynamics at high throughput. Meta-analysis of the quantitative variates extracted from HCS-CLM will contribute to a better biological understanding of biofilm traits.