The diketopiperazines (DKPs)-containing natural products represent a large class of secondary metabolites mainly produced by bacteria, fungi, marine invertebrates and higher organisms. DKPs were biosynthesized by condensation of two amino acids including tryptophan, proline, histidine and phenylalanine. Interest in DKPs is due to their activities in various pharmacological assays ranging from antibacterial, antifungal, antiviral to immunosuppressive. In recent studies, the nocazines are a newly defined family of antibacterial and cytotoxic cyclic dipeptides produced by Nocardiopsis dassonvillei and N. alba. Our investigation with a deep-sea sediment strain, YIM M13066, identified as a Nocardiopsis sp., was found to produce six DKPs, including two new DKPs, nocazines F (1) and G (2), and four known DKPs (3–6). In this study, the fermentation, isolation, and structural elucidation and bioactivity of compounds 1–6 from Nocardiopsis sp. YIM M13066 were described. In summary, the chemical study on strain YIM M13066 led to the isolation and characterization of six DKPs. Compared with known nocazines, nocazines F (1) and G (2) showed a broad spectrum of cytotoxicity against the human tumor cell lines, which suggested that the O-methyl group at C-5, 18-OH group and the Δ6(7) double bond may be the key active groups of nocazines. Besides, nocazine F exhibited a strong inhibition on the T3SS of S. enterica serovar Typhimurium, which could be a promising candidate for a novel type of antibiotics.