Recent and ongoing studies evidence that certain LED lights, suitable for illuminating the urban fabric, avoiding primary colours such as blue, green, and red, may halt the biological colonization on stone monuments, mainly caused by algae and cyanobacteria forming subaerial biofilms (SABs). However, the light-triggered mechanisms that cause changes in the growth of the phototrophic species is so far unknown. An environmental proteomic approach could shed light on this aspect, providing information on the changes on SAB’s metabolism under the stress inflicted by different ornamental lighting. Here, laboratory-made SABs mainly composed of Chlorophyta, and at lesser extent Streptophyta and Cyanophyta, were subjected to three lighting conditions for monuments: cool white, warm white and, amber+green (with biostatic effect and under trial) A control without light (i.e., darkness) was also included for comparison. Cool white and warm white illumination severely affected SAB’s proteome reducing their total peptide identifications being the extent of this reduction dependent on the organisms’ genera. The photo-damaging effect of amber+green on the biofilm metabolism was clarified, revealing its negative impact on the photosystems I and II and antenna pigments production, and its triggering effect on the protein metabolism (synthesis, folding, and degradation). This research provides for the first-time a thorough description of the proteomic changes induced by lighting on SABs colonising illuminated monuments in urban areas. The insights gained will help to better design the lighting of monuments, with the inclusion of ornamental lighting as one of the elements to be taken into account in the preventive conservation of the built heritage.