We investigated the incorporation of nitrogen from amino acids and urea by the microbial community in intertidal surface sediment, focusing on the relative contributions of bacteria versus algae to total microbial nitrogen incorporation and the (un)coupled incorporation of carbon from these organic substrates. Dual-labeled (15N and 13C) urea and an amino acid mixture were added to surface sediment from 2 intertidal mudflats in the Scheldt Estuary (The Netherlands), and 15N and 13C were subsequently traced into bulk sediment, total hydrolysable amino acids (THAAs) and the bacterial biomarker d-alanine (d-Ala) over a 24 h incubation period. All added 15N from urea and the amino acids was incorporated into microbial biomass within 24 h, with relatively rapid incorporation of 15N from the amino acids. The bacterial contribution to total microbial 15N incorporation (derived from 15N incorporation into d-Ala) was large during the first 1 to 2 h of incubation, but small to negligible after 24 h for both substrates, indicating that total 15N incorporation was dominated by benthic microalgae (diatoms) that also dominated total microbial biomass in the sediment. Comparison of 15N versus 13C incorporation into total microbial biomass (THAAs) after 24 h showed strong preferential incorporation of urea-N over urea-C. Incorporation of nitrogen and carbon from the amino acids was partially uncoupled, indicating that a large fraction (≥50%) of amino acid-N was taken up as NH4+ resulting from extracellular amino acid oxidation.