The red swamp crayfish, Procambarus clarkii, and the golden apple snail, Pomacea canaliculata, are globally widespread invasive species that can cause serious damage to aquatic ecosystems in the invaded areas. However, the impact of their co-invasion on native ecosystems has only rarely been studied. To explore the niche competition between co-invasive and native species, we investigated a natural pond that was co-invaded by P. clarkii and P. canaliculata. We used a fatty acid biomarker technique to analyze differences in feeding habits, niche widths, and trophic levels among P. clarkii, P. canaliculata, and the native snail Bellamya aeruginosa. We also conducted indoor predation control experiments to verify that the alien species directly prey on B. aeruginosa. Principal component analysis (PCA) of fatty acids in muscles revealed larger areas of the confidence ellipses in P. clarkii and P. canaliculata compared to B. aeruginosa, indicating that the alien species had broader niche widths and superior trophic resource exploitation. Also, the percentage content of C18:1 n-9 + C22:6 n-3 was significantly higher in P. clarkii than in P. canaliculata and B. aeruginosa, suggesting that the crayfish is more carnivorous. C15 + C17 content was significantly higher in B. aeruginosa than in the alien species, suggesting bacteria as the native snail"s main carbon source. In contrast, C18:2 + C18:3 content was significantly higher in the alien species, demonstrating that their main carbon source is vascular plant material. Finally, the ratio of polyunsaturated to saturated fatty acids (PUFA/SFA) was also higher in the alien species, indicating that they reside at a higher trophic level and exhibit stronger competitive abilities compared to B. aeruginosa. Our indoor predation experiments showed that P. clarkii significantly more often preyed on juvenile B. aeruginosa than on juvenile P. canaliculata. We also observed predation of adult P. canaliculata on juvenile B. aeruginosa, albeit at a lower rate. In summary, by combining the results of fatty acid biomarker techniques and indoor predation control experiments, we demonstrate that both the niche width and the trophic level of P. clarkii are higher than those of P. canaliculata, while P. canaliculata exhibits significantly broader niche widths and higher trophic positions comparing to the native species B. aeruginosa. The co-invasion of these two species thus may have a significant negative impact on native snails.