Expansins cause plant cell wall loosening and are present primarily in the plant kingdom. Gene sequence analysis suggests that expansins are present in several plant-colonizing or plant-pathogenic bacteria and fungi. However, experimental evidence of microbial expansin activity is largely lacking. Here we provide evidence that expansins from three plant pathogenic bacteria and one fungus cause extension of cell walls in vitro and weaken filter paper networks, without lytic activity. Since expansins were able to weaken cellulose networks, we tested whether they synergistically enhanced the activity of several cellulases in hydrolysis of cellulose. The microbial expansins did not show such synergism beyond the nonspecific effect of bovine serum albumin. Our results show that the expansins present in several pathogenic microbes have weak wall-loosening activity and we infer a role for these expansins in plant pathogenesis. Additionally, the convenient expression of several expansins in Escherichia coli makes a future comparative structure-function analysis among expansins possible in order to understand their activity at the molecular level.