Analysis of the composition of the marine-dissolved organic matter has highlighted the importance of D-amino acids, whose origin is attributed mainly to the remains of bacterial peptidoglycan released as a result of grazing or viral lysis. However, very few studies have focused on the active release of D-amino acids by bacteria. With this purpose, we measured the concentration of dissolved amino acids in both enantiomeric forms with two levels of complexity: axenic cultures of Vibrio furnissii and Vibrio alginolyticus and microcosms created from marine microbial assemblages (Biscay Bay, Cantabrian Sea) with and without heterotrophic nanoflagellates (HNFs). Axenic cultures showed that only D-Ala was significantly released and accumulated in the medium up to a concentration of 120 nM, probably as a consequence of the rearrangement of peptidoglycan. The marine microbial assemblages showed that only two D-amino acids significantly accumulated in the environment, D-Ala and D-aspartic acid (Asp), in both the absence and presence of HNFs. The D/L ratio increased during the incubation and reached maximum values of 3.0 to 4.3 for Ala and 0.4 to 10.6 for Asp and correlated with prokaryotic and HNF abundance as well as the rate of prokaryotic thymidine and leucine incorporation. Prokaryotes preferentially consumed L-amino acids, but the relative uptake rates of D-Ala significantly increased in the growth phase. These results demonstrate that bacteria can release and consume D-amino acids at high rates during growth, even in the absence of viruses and grazers, highlighting the importance of bacteria as producers of dissolved organic matter (DOM) in the sea.