Calcium ions (Ca2+) are known to enhance biofilm structural integrity in many bacteria, including Pseudomonas aeruginosa, by cross-linking exopolysaccharides. Still, their role in the soil bacterium P. putida remains largely unexplored. Here, we investigated the dose-dependent effects of calcium chloride on P. putida KT2442 biofilm architecture, matrix properties, nanomechanics, and global gene expression. Contrary to the stabilizing role observed in P. aeruginosa, calcium induced complex biphas