The efficiency of six different cleaning protocols was studied and quantified by measuring ATP and carbohydrate (CH) concentrations. The detailed characterization of changes in the biofilm structure due to the application of various cleaning agents was performed by means of confocal laser scanning microscopy (CLSM). Biofilm samples were cultivated over a period of 28 days under well-defined conditions in space and time to mimic seriously biofouled membrane surfaces which were tested under reproducible conditions. Quantitative structural parameters were calculated from CLSM image stacks. Imaging data revealed visually and quantitatively differences at the microscale in the behavior of cleaning procedures applied. Moreover, these data showed correlations with quantitative biomass parameters ATP and CH at the macroscale. Hence, this study supports the use of CLSM as a research tool to understand in detail the effects of selected cleaning agents on biofilm structure and presents supportive data for the efficacy of certain cleaning protocols. (C) 2013 Elsevier B.V. All rights reserved.