Environmental DNA (eDNA) is a very promising approach to facilitate and improve the aquatic species monitoring, which is crucial for their management and conservation. In comparison with the plethora of monitoring studies in the fields, relatively few studies have focused on experimentally investigating the “ecology” of eDNA, in particular pertaining to processes influencing the detection of eDNA. The paucity of knowledge about its ecology hampers the use of eDNA analysis to its full potential. In this study, we experimentally evaluated the impact of several biotic and abiotic factors on the rate of production and degradation of eDNA. Individuals of three freshwater fish species (brown bullhead, tench, and yellow perch) with distinct ecology were placed in two types of water from the St. Lawrence River (Québec, Canada) with very distinct physicochemical characteristics and at three different temperatures. Water samples were then filtered at predetermined time intervals, and quantitative PCR was used to quantify the eDNA in each sample. We found that temperature, species, water types, and some interactions between these factors had a strong effect on the production and degradation of eDNA. The results of this study enhance our knowledge about the ecology of eDNA, thus improving eDNA data interpretation.