Environmental DNA (eDNA) has rapidly emerged as a promising biodiversity monitoring technique, proving to be a sensitive and cost‐effective method for species detection. Despite the increasing popularity of eDNA, several questions regarding its limitations remain to be addressed. We investigated the effect of sampling me‐ dium and time, and preservation methods, on fish detection performance based on eDNA metabarcoding of neotropical freshwater samples. Water and sediment sam‐ ples were collected from 11 sites along the Jequitinhonha River, Southeastern Brazil; sediment samples were stored in ethanol, while the same amounts of water per sam‐ ple (3 L) were stored in a cool box with ice, as well as by adding the cationic surfactant benzalkonium chloride (BAC). Sediment and water samples yielded a similar amount of fish MOTUs (237 vs. 239 in the first sampling event, and 153 vs. 142 in the second sampling event). Water stored in ice provided better results than those preserved in BAC (239 and 142 vs. 194 and 71 MOTUs). While documenting the effectiveness of eDNA surveys as practical tools for fish biodiversity monitoring in poorly accessible areas, we showed that keeping water samples cooled results in greater eDNA recovery and taxon detection than by adding cationic surfactants (BAC) as sample pre‐ servatives. Furthermore, by comparing two sets of samples collected from the same locations at a 3‐week interval, we highlight the importance of conducting multiple sampling events when attempting to recover a realistic picture of fish assemblages in lotic systems.