- Bacterial biofilms are integrated, single‐ or multi‐species communities of cells that play profound roles in human health and disease. The formation of biofilms requires interactions between bacteria and the surfaces they colonize, and the surface can specifically impact the structure, function, and composition of these communities. Investigating biofilm formation in situ, their assembly kinetics, and particularly identifying substances that could interfere with or inhibit biofilm growth is thus a major scientific and practical goal. It is shown that thin dip‐coated films comprising a transparent sol–gel framework and polydiacetylene, a unique conjugated polymer that can undergo color and fluorescence transitions, both promote rapid growth of bacterial biofilms as well as allow colorimetric and fluorescence detection of biofilm formation. Microscopy data demonstrate that the bacterial cells and resultant biofilm specifically target the polydiacetylene domains embedded within the silica‐gel matrix, consequently inducing dramatic colorimetric and fluorescence transitions. The mesoporous silica/polydiacetylene matrix can further host other chemical substances allowing evaluation of their biofilm inhibitory effects through simple chromatic screening. Overall, the polydiacetylene/sol–gel films constitute a novel generic platform for promoting bacterial biofilms and their in situ analysis.