This study conducted several approaches to determine development and succession of different types of biofilms (i.e., colonization and accumulation biofilms). Changes in total metabolic activity, bacterial and algal composition within different biofilms from two river ecosystems were analyzed. They were related to water quality parameters in order to assess major factors influencing biofilm growth. Significant differences in chlorophyll a concentrations in biofilms and water between two rivers were due to differences in light intensity, water current velocity, and turbidity. Colonization of epilithic algae in biofilms mainly resulted from high levels of nutrients (up to 2.8 mg L−1 of phosphate) and water chlorophyll a, and may be caused by attachment of planktonic algae. However, epilithic algae may also serve as the source of planktonic algae. Oxidizable substrates measured as chemical oxygen demand were found to directly increase bacterial growth or indirectly affect growth a week later. One-month colonization biofilms were the most sensitive to change of water quality, and had the greatest number of significant relationships to physico-chemical and biological parameters among three types of biofilms. This suggested that 1-month colonization biofilms were applicable for biomonitoring water quality.