Declining biodiversity is a growing crisis globally, and the field of conservation is heavily reliant on species monitoring and resultant population data. However, these data are challenging to collect and require intensive sampling along with extensive taxonomic expertise. Environmental DNA (eDNA) is an efficient and non-invasive method of monitoring organisms in aquatic habitats. However, critical knowledge gaps exist regarding controls on eDNA removal from the water column, especially in flowing waters (i.e., streams and rivers). Moreover, allochthonous organic matter inputs via autumn leaf fall is an important seasonal change in stream ecosystems, especially in smaller, headwater streams, and leaf litter accumulation (i.e., debris dams) and decomposition may modify biotic and abiotic conditions that influence eDNA retention in streams. We conducted experimental releases of Common Carp (Cyprinus carpio) and Steelhead Trout (Oncorhynchus mykiss) eDNA in recirculating mesocosms (n=24) to quantify eDNA removal rates in conjunction with varying densities and decomposition stage of submerged leaf litter. We found that leaf density had no significant effect on eDNA removal rates, but removal rates for smaller eDNA particles (0.4 μm) were 74% higher in mesocosms with biofilm-colonized leaves relative to those with “fresh” uncolonized leaves (Tukey-Kramer Test, p < 0.001). In contrast, we found no significant difference in removal rates between colonized and uncolonized leaves for larger eDNA particles (1.2 μm). Overall, eDNA removal rates were higher for smaller eDNA particles, but only in the presence of biofilm-colonized leaves, suggesting a biological mechanism for eDNA removal from the water column. These results suggest that seasonal leaf litter plays a role in the fate of eDNA particles, which is mediated by eDNA particle size and leaf litter quality. Understanding controls on eDNA removal has the potential to assist in the interpretation of eDNA data in natural systems.