- Mesostructured silica films have attracted interest as potential platforms for sensing, molecular sieving, catalysis, and others. The fabrication of free-standing silica films on water, however, is challenging due to the need for scaffolding agents that would constitute effective templates. We describe the assembly of thin film at the air/water interface comprising quaternary silicates and polydiacetylene (PDA), a unique chromatic polymer forming two-dimensional conjugated networks, and the use of these films for biological sensing. PDA exhibits a dual role in the system—both as the amphiphilic matrix facilitating immobilization of the silicate colloidal units at the air/water interface and additionally a chromatic reporter that undergoes visible blue–red transitions, accompanied by fluorescence transformations, in the presence of analytes. We demonstrate the application of the silicate/PDA thin films for the detection of bacterial proliferation.