<p dir="ltr"><i>Pseudomonas aeruginosa</i> (<i>P. aeruginosa</i>) is a Gram-negative bacterium responsible for many infections in immunocompromised humans. This multi-drug resistance human pathogen can form biofilms, which help protect it from not only clinical treatment but also from main homeostasis and metabolism. Understanding biofilm structures is critical to help combat biofilm formation and develop better ways to treat <i>P. aeruginosa</i> infections. A molecule that helps biofilm formation and virulence infections for <i>P. aeruginosa</i> is pyocyanin, which is believed to be correlated with the invasiveness of the bacteria and the stabilization of biofilms. To better understand the role of pyocyanin in assisting <i>P. aeruginosa</i> with survival, we applied optical imaging to study pyocyanin in biofilms and under blue light treatment. Using nonlinear optical imaging methods, we could successfully detect the aggregation of pyocyanin in biofilms. Furthermore, we discovered that pyocyanin protects <i>P. aeruginosa</i> from blue light inactivation. In addition, we found that blue light treatment alters the structure of pyocyanin, leading to irreversible changes that produce distinct spectra in UV-Vis and fluorescence signals. <i>These results provide new insights into how pyocyanin protects </i><i>P. aeruginosa</i> in blue light treatment. Further investigation would lead to better treatment strategies for more effective treatment of <i>P. aeruginosa</i> and biofilms for various infections.</p>