Three new emestrin-type thiodiketopiperazines including didethio-11a-methylthioemestrin (1), 7′-epi-didethio-11a-methylthioemestrin (2), and 2″-desmethyl-MPC1001F (3), along with three known analogues emestrin (4), dethiosecoemestrin (5), and emestrin H (6), were isolated and identified from the culture extract of Aspergillus nidulans SD-531, a fungus obtained from the deep-sea cold seep sediment sample collected from the South China Sea. Their structures and absolute configurations were determined by spectroscopic analysis and TDDFT-ECD calculations. Compounds 3–6 showed antimicrobial activity against some of the tested strains, while compounds 3–5 had cytotoxic activity against the tumor cell line Huh 7.5. Compound 4 exhibited the strongest cytotoxic activity with an IC₅₀ value of 0.25 μM, which was better than that of the positive control sorafenib.