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ABSTRACT Recent research into the spatiotemporal dynamics of eDNA in marine environments indicates that eDNA signals are highly localized and may dissipate beyond detection levels within a few hours of production. This affects whether single‐timepoint eDNA sampling, which generally occurs during daylight hours, or cyclic (day/night) interval eDNA sampling is necessary to detect both diurnal and nocturnal marine species. Our study investigated short‐term variability in eDNA derived from fishes and macroinvertebrates across three temperate reef sites in eastern Tasmania, Australia. Simultaneous eDNA and underwater visual census (UVC) diver transect surveys were conducted every 6 h over a 24‐h period to investigate whether eDNA was able to detect marine species outside of their UVC‐observed diel activity. We report that single‐timepoint eDNA sampling can detect both diurnal and nocturnal species on temperate reefscapes. A lack of eDNA compositional turnover between day and night suggests that eDNA persists beyond 12 h and/or is continuously produced by both diurnal and nocturnal reef taxa, irrespective of diel behavioral changes observed by UVC. Given high eDNA sample variability, however, we recommend a high replication level (> 10 × 1 L samples) to produce robust site community composition profiles. This study builds on emerging literature on short‐term variability in eDNA, assisting in the design of future eDNA studies at sites with pronounced variation in faunal activity between day and night.