Fouling is a fundamental problem in membrane processes, and biofouling (i.e. fouling which is caused by microorganisms) control is considered a major challenge in operating membrane systems. For more efficient control and prediction of biofouling of membranes in drinking water treatment, assays which indicate the occurrence of biofouling need to be developed. Such assays can also provide a fundamental understanding of mechanisms of biofouling. For this it is necessary to study the groups of microorganisms involved in different stages of membrane biofouling. The composition of biofilm microbial communities on membrane surfaces can be analyzed using culture independent microscope- and molecular-based methods. In this study we operated a hollow fiber, microfiltration (MF) membrane test unit that was fed with tap water enriched with natural organic material (humus) before ozonation and biooxidation. The confocal laser scanning microscopy (CLSM) analyses clearly showed that the biofilm gradually developed on the membrane surfaces with time, which had a strong positive correlation with the increase in transmembrane pressure. This indicated that the biofilm formation induced the membrane fouling. Initial examination of the biofouling layer on membranes in drinking water was carried out using DNA probes specific for alfa, beta and gamma subclasses of proteobacteria as well as a probe for Eubacteria. As model bacteria S. paucimobilis (alfaproteobacteria), Burkholderia cepacia (beta-proteobaceria) and Legionella pneumophila (gamma-proteobacteria) were used as these bacteria are common in water distribution systems. Agarose coating of the samples was introduced as repulsion between sample, probe and microscope slide was observed. The proportions of alfa-, beta- and gammaproteobacteria were 14%, 6% and 2%, respectively, in suspension of 17 day old biofouling on hollow fiber membrane. The total bacterial number in samples (30 ul) was 106.