Invasive macrophyte can significantly influence riverine fishes by changing the biotic and abiotic environment. It is important to understand differences in responses of fishes to invasive macrophyte in order to predict the effects of invasive macrophyte overgrowth on fish assemblage structure in riverine systems. This study examined the relationships between the coverage area of invasive macrophyte, Egeria densa, and fish assemblage compositions in the Gonokawa River in Japan on September and November in 2020 using quantitative environmental DNA metabarcoding. Our results indicate that the species-habitat relationships varied among the fishes and between the seasons in the study sites. Four fishes that tend to use aquatic vegetations as their habitats (e.g., Coreoperca kawamebari) were significantly and positively associated with the upstream coverage area of E. densa in the study sites. In contrast, fishes that prefer sand and gravel substrates (e.g., Pseudogobio esocinus) had significant negative correlations with the coverage area of E. densa. Further, the correlations between these fishes and the upstream coverage area in November (abundance peak of E. densa) were higher than those in September. These results suggest that the overgrowth of the invasive macrophyte would greatly change the fish assemblage compositions in the riverine system.