The goal of this comparative study was to investigate biofilm forming microorganisms living in washing machines (WMs). Biofilms were sampled from 11 washing machines from four countries and three continents. Among the 94 isolated strains, 30% were potential human pathogens. Representative strains were selected and biofilm formation was evaluated with the crystal violet (CV) assay. The majority of the WM isolates formed more biofilm than their reference strains. Biofilms of P. putida WM (the largest biofilm producer) were exposed to different concentrations (0.0007-7 g l(-1)) of the standard detergent IEC-A* at 30°C for 30 min and observed with confocal laser scanning microscopy. Using quantitative CVA, P. putida WM biofilm removal required higher detergent concentrations than the type strain. However, for both strains the recommended detergent concentration (7 g l(-1)) was insufficient to completely clean surfaces from cell debris and exopolymeric substances.