<jats:title>Abstract</jats:title><jats:p>Two new antibacterial thiodiketopiperazine derivatives (TDKPs), 7‐dehydroxyepicoccin H and 7‐hydroxyeutypellazine F, along with seven known TDKP analogs, were isolated and identified from <jats:italic>Epicoccum nigrum</jats:italic> SD‐388, a deep‐sea‐sediment‐derived fungus. The structures of these compounds were elucidated on the basis of detailed spectroscopic analysis. The absolute configuration of 7‐dehydroxyepicoccin H was established by X‐ray crystallographic analysis, while 7‐hydroxyeutypellazine F was determined by ECD experiment and TDDFT‐ECD calculation. The antibacterial activities against human and aquatic pathogens were evaluated. 7‐Dehydroxyepicoccin H and 7‐hydroxyeutypellazine F displayed inhibitory activities against aquatic pathogens <jats:italic>Vibrio vulnificus</jats:italic>, <jats:italic>V. alginolyticus</jats:italic>, and <jats:italic>Edwardsiella tarda</jats:italic>, with MIC values ranging from 4.0 to 8.0 μg/mL.