Ten new and 13 known prenylated 2,5-diketopiperazines (2,5-DKPs) were identified from co-culture of Penicillium sp. DT-F29 with Bacillus sp. B31. The constitutions and relative configurations of new compounds were established by 1D and 2D NMR spectroscopic data analysis. The isolated 2,5-DKPs in bioactivity test showed bromodomain-containing protein 4 (BRD4) inhibitory activities, and compound 14, with IC50 values of 6.54 μM, was potential for BRD4 inhibitor discovery.