Microplastic pollution and ingestion are ubiquitous phenomena in freshwater ecosystems. However, our understanding of the role of trophic niche in microplastic ingestion is still limited. Here, we quantified the level of microplastic (700 μm to 5 mm) contamination for macroinvertebrates and fish within the Garonne river. We then used stable isotope analyses (δ13C and δ15N) to quantify trophic niches. We first demonstrated that the abundance of ingested microplastics differed between macroinvertebrates and fish and was not significantly related to microplastic pollution. We then found that microplastic characteristics (shape, color, size, and polymer composition) differ between the abiotic (surface waters and sediments) and biotic (ingested by macroinvertebrates and fish) compartments. The abundance of ingested microplastics increased with the size of organisms in both fish and macroinvertebrates and tended to increase with trophic position in macroinvertebrates only. Finally, the origin of the resources consumed by fish significantly affected the abundance of microplastics ingested. Altogether, these results suggest the absence of microplastic bioaccumulation in freshwater food webs and the dominance of direct consumption, most likely accidentally. The use of stable isotope analyses is therefore crucial to improve our understanding of microplastic ingestion by wild organisms.