Extracellular enzymes are major drivers of biogeochemical nutrient and carbon cycling in surface water. While photoinactivation is regarded as a major inactivation process of these enzymes, the underlying molecular changes have received little attention. This study demonstrate how light exposure leads to a rapid loss of phosphatase, aminopeptidase and glucosidase activities of biofilm samples and model enzymes. Here, an optimized proteomics approach allowed simultaneous observation of inactivation and molecular changes. Site-specific fingerprints of degradation kinetics have been generated and visualized in the three-dimensional proteins. Oxidation of tryptophan, the chromophoric target, initiated secondary reactions. Evidence was obtained that tyrosine residues act as intramolecular antioxidants, reflected in decelerated decay of tryptophan- and enhanced decay of tyrosine-containing peptides. In addition, subsequent methionine oxidation and disulfide reduction contribute to heterogeneous photodamage. The proximity to tryptophan residues explains >95% of the photodamage across the protein structures. The presence of redox-active organic matter or a model antioxidant in solution quenched not only photoinactivation and tryptophan oxidation but also all subsequent damage. The developed analytical approach can be applied to other research questions in environmental sciences where site-specific damage in a protein is essential.