Biofilm formation on membrane surfaces causes many operational problems such as a decrease in permeate flux and an increase in hydraulic resistance. In this study, the ability of bacteria to pass through microfiltration (MF) membranes and the growth potential of microfilterable bacteria were investigated in order to understand biofouling in MF-reverse osmosis (RO) integrated membrane systems. Growth of microfilterable bacteria in MF permeate was observed, indicating that not all MF membranes can guarantee the total rejection of bacteria. Changes in natural organic matter (NOM) characteristics and growth potential of bacteria during the treatment process are important factors in the occurrence of biofilm development in water treatment systems. Analysis of protein-like and humic-like substances in NOM of two successive RO stages revealed an increase in the concentrations of both biopolymers and humic substances of RO concentrates. Unexpectedly, the use of antiscalants was seen to enhance the growth of bacteria in the RO feed water in this study. Bacterial 16s rRNA pyrosequencing revealed that passing source water through the MF membranes dramatically changed bacterial community structure. The bacterial communities that passed through the MF steps primarily belonged to the family Comamonadaceae. However, several bacteria groups including Flavobacteriaceae, Sphingobacteriaceae and Sphingomonadaceae selectively composed the biofilm community formed on the RO membranes. Thus, understanding the selectivity and filterability of MF towards microorganisms involved in biofouling on RO membrane surfaces is crucial for the improvement of membrane-related operational processes.