Use of environmental DNA (eDNA) to assess distributions of aquatic and semi-aquatic macroorganisms is promising, but sampling schemes may need to be tailored to specific objectives. Given the potentially high variance in aquatic eDNA among replicate grab samples, compositing smaller water volumes collected over a period of time may be more effective for some applications. In this study, we compared eDNA profiles from composite water samples aggregated over three hours with grab water samples. Both sampling patterns were performed with identical autosamplers paired at two different sites in a headwater stream environment, augmented with exogenous fish eDNA from an upstream rearing facility. Samples were filtered through 0.8 μm cellulose nitrate filters and DNA was extracted with a cetyl trimethylammonium bromide procedure. Eukaryotic and bacterial community profiles were derived by amplicon sequencing of 12S ribosomal, 16S ribosomal, and cytochrome oxidase I loci. Operational taxa were assigned to genus with a lowest common ancestor approach for eukaryotes and to family with the RDP Classifier software for prokaryotes. Eukaryotic community profiles were more consistent with composite sampling than grab sampling. Downstream, rarefaction curves suggested faster taxon accumulation for composite samples, and estimated richness was higher for composite samples as a set than for grab samples. Upstream, composite sampling produced lower estimated richness than grab samples, but with overlapping standard errors. Furthermore, a bimodal pattern of richness as a function of sequence counts suggested the impact of clumped particles on upstream samples. Bacterial profiles were insensitive to sample method, consistent with the more even dispersion expected for bacteria compared with eukaryotic eDNA. Overall, samples composited over 3 h performed equal to or better than triplicate grab sampling for quantitative community metrics, despite the higher total sequencing effort provided to grab replicates. On the other hand, taxon-specific detection rates did not differ appreciably and the two methods gave similar estimates of the ratio of the common fish genera Salmo and Coregonus at each site. Unexpectedly, Salmo eDNA dropped out substantially faster than Coregonus eDNA between the two sites regardless of sampling method, suggesting that differential settling affects the estimation of relative abundance. We identified bacterial patterns that were associated with eukaryotic diversity, suggesting potential roles as biomarkers of sample representativeness.