Carbon and nitrogen stable isotope ratios (δ¹³C and δ¹⁵N) have been utilized as powerful tools for tracing energy or material flows within food webs in a range of environmental studies. However, the techniques have rarely been applied to the study of biological wastewater treatment technologies. We report on the spatial changes in δ¹³C and δ¹⁵N in sludge and its associated biotic community in a wastewater treatment system. This system consisted of an upflow anaerobic sludge blanket (UASB) and a down-flow hanging sponge (DHS) which is a novel type of trickling filter. The results showed clear spatial changes in the δ¹³C and δ¹⁵N of suspended solids (SS), retained sludge, and macrofauna (oligochaetes and fly larvae) in the system. The δ¹³C and δ¹⁵N was used as a natural tracer to determine the SS dynamic throughout the system. The results imply that SS in the DHS effluent was mainly eluted from the retained sludge in the lower section of the DHS reactor. The δ¹⁵N of the retained sludge in the DHS reactor increased drastically from the inlet towards to the outlet, from -0.7‰ to 10.3‰. This phenomenon may be attributed to nitrogen conversion processes (i.e. nitrification and denitrification). The δ¹⁵N of oligochaetes also increased from the inlet to the outlet, which corresponded well to that of the retained sludge. Thus, the δ¹⁵N of the oligochaetes might simply mirror the δ¹⁵N of the retained sludge. On the other hand, the δ¹³C and δ¹⁵N of sympatric fly larvae differed from those of the oligochaetes sampled, indicating dietary differences between the taxa. Therefore δ¹³C and δ¹⁵N reflected both treatment and dietary characteristics. We concluded that δ¹³C and δ¹⁵N values are potentially useful as alternative indicators for investigating microbial ecosystems and treatment characteristics of biological wastewater treatment systems.