The use of environmental DNA (eDNA) to determine the presence and distribution of aquatic organisms has become an important tool to monitor and investigate freshwater communities. The successful application of this method in the field, however, is dependent on the effectiveness of positive DNA verification, which is influenced by site-specific environmental parameters. Factors affecting lower eDNA concentrations in aquatic ecosystems include flow conditions, and the presence of substances that possess DNA-binding properties or inhibitory effects. In this study we investigated the influence of different environmental parameters on the detection success of eDNA using the invasive goby Neogobius melanostomus. In a standardized laboratory setup, different conditions of flow, sediment-properties, and fish density were compared, as well as different potential natural inhibitors such as algae, humic substances, and suspended sediment particles. The presence of sediment was mainly responsible for lower eDNA detection in the water samples, regardless of flow-through or standing water conditions and a delayed release of eDNA was detected in the presence of sediment. Humic substances had the highest inhibitory effect on eDNA detection followed by algae and siliceous sediment particles. The results of our study highlight that a successful application of eDNA methods in field surveys strongly depends on site-specific conditions, such as water flow conditions, sediment composition, and suspended particles. All these factors should be carefully considered when sampling, analyzing, and interpreting eDNA detection results.