Bacterial biofilms may cause chronic infections due to their ability to evade clearance by the immune system and antibiotics. The persistent biofilms induce a hyperinflammatory state that damages the surrounding host tissue. Knowledge about the components of biofilms that are responsible for provoking the harmful but inefficient immune response is limited. Flagella are known to stimulate the response of polymorphonuclear leukocytes (PMNs) to planktonic solitary bacteria. However, we provide evidence that flagella are not a prerequisite for the response of PMNs to Pseudomonas aeruginosa biofilms. Instead, we found that extracellular matrix polysaccharides in P. aeruginosa biofilms play a role in the response of PMNs toward biofilms. Using a set of P. aeruginosa mutants with the ability to produce a subset of matrix exopolysaccharides, we found that P. aeruginosa biofilms with distinct exopolysaccharide matrix components elicit distinct PMN responses. In particular, the PMNs respond aggressively toward a biofilm matrix consisting of both Psl and alginate exopolysaccharides. These findings are relevant for therapeutic strategies aimed at dampening the collateral damage associated with biofilm-based infections.