Widespread introductions of non-native species, including aquaculture and ornamental species, threaten biodiversity and ecosystem functioning by modifying the trophic structure of communities. In this study, we quantified the multiple facets of trophic disruption in freshwater communities invaded by Nile tilapia, by comparing uninvaded and invaded rivers downstream of the Pearl River, China. Nile tilapia invasion reduced the trophic status of native fish species by forcing native herbivores and planktivores to seek new food sources. The food chain was also shortened by decreasing the trophic levels of native invertivores, omnivores, and piscivores, while the total isotopic niche area (TA) of native invertivores, omnivores, piscivores, and planktivores species also decreased. Simultaneously, Nile tilapia invasion affected the isotopic diversity of the fish community. Decreasing isotopic richness (IRic), isotopic evenness (IEve), and increasing isotopic uniqueness (IUni) indicated that Nile tilapia had a high trophic niche overlap with native species and competed with native species for food resources, and even caused the compression of the trophic niche of native species. Understanding the process described in this study is essential to conserve the stability of freshwater ecosystems, and improve the control strategy of alien aquatic organisms in south China.