Environmental management and understanding of ecosystems requires accurate assessment of biodiversity. Environmental DNA (eDNA) offers a non-invasive and objective biodiversity survey method, focusing on the detection of free cellular material. Despite significant advantages over traditional methods many of the dynamics of eDNA within the water column are still unknown such as the persistence and transport of molecules. So far, most eDNA studies have focused on detecting species in water samples. This study focuses on biofilms which have never previously been used to detect eDNA and offer a novel sampling medium for future studies using eDNA. This study aims to detect macrobial eDNA within freshwater biofilms, model the uptake and persistence of eDNA within biofilms over time and determine environmental factors that affect the spatio-temporal persistence of eDNA. DNA of mayfly (Ephemera danica), Daphnia (D. magna) and European eel (Anguilla anguilla) was added to four experimental mesocosms of lotic freshwater, each associated with different land-use types and pH. Biofilms were collected over 43 hours and eDNA quantified using qPCR with CO1 primers. Only mayfly DNA was detected within the biofilm. The quantity of mayfly DNA had a strong negative relationship with time and a positive association with pH, demonstrating that eDNA degraded quickly over time and faster in mesocosms with lower pH. In conclusion, this study builds on the wider knowledge of how eDNA persists in lotic freshwater and provides a new sampling medium for further eDNA studies.