Triclosan (TCS, 5-chloro-2-(2,4-dichlorophenoxy) phenol) is a broad-spectrum antimicrobial compound. Owing to its wide use, TCS has been frequently detected in river systems, especially in the (sub-)tropics. However, little information on its interaction with river biofilm in the (sub)tropics is currently available. In the present study, subtropical river biofilms were chronically exposed to TCS for 14 d at concentrations of 0.1–100 μg/L in artificial river water, which was followed by a 7 d recovery period. The results show that 100 μg/L TCS inhibited the growth of river biofilms and the no-observed-effect concentration (NOEC) of TCS on river biofilms was 10 μg/L. The affected biofilms did not completely recover within the 7 d of recovery period due to the adsorbed TCS which was not removed together with dissolved TCS. Exposure to TCS caused significant changes in prokaryotic species composition of river biofilms but no significant effects on eukaryotic species composition. In particular, the relative abundance of several TCS-tolerant bacterial species (e.g., Pseudoxanthomonas mexicana, Sphingopyxis alaskensis and Sphingomonas wittichii) in river biofilms increased following exposure to 10 and 100 μg/L TCS. River biofilm efficiently removed TCS from the liquid phase and the pH values of the aquatic system significantly affected the removal efficiency of TCS (from 36% at pH 6.5 to 60% at pH 8.5). No degradation products were detected in the liquid phase after 5 days of exposure, possibly due to strong adsorption of the hydrophobic degradation products to river biofilms and through biodegradation by bacteria utilizing TCS and its degradation products as source of carbon and energy for growth, such as Methyloversalitis universalis and Methylobacterium aquaticum.