Dissolved organic nitrogen (DON) acts as a large reservoir of fixed nitrogen. Whereas DON utilization is common in the microbial community, little is known about utilization by macrophytes. We investigated the ability of the coexisting temperate marine macrophytes Zostera noltii, Cymodocea nodosa, and Caulerpa prolifera to take up nitrogen and carbon from small organic substrates of different molecular complexities (urea, glycine, L-leucine, and L-phenylalanine) and from dissolved organic matter (DOM) derived from algal and bacterial cultures (substrates with a complex composition). In addition to inorganic nitrogen, nitrogen from small organic substrates could be taken up in significant amounts by all macrophytes. Substrate uptake by the aboveground tissue differed from that of the belowground tissue. No relationships between carbon and nitrogen uptake of small organics were found. The preference for individual organic substrates was related to their structural complexity and C:N ratio. Uptake of algae-derived organic nitrogen was of similar magnitude as inorganic nitrogen, and was preferred over bacteria-derived nitrogen. These results add to the growing evidence that direct or quick indirect DON utilization may be more widespread among aquatic macrophytes than traditionally thought.