Two known naphthocoumarins, chrysomycins A (1) and B (2), along with one new naphthocoumarin chrysomycin C (3) were isolated from the antimicrobial strain of Streptomyces sporoverrucosus (MTCC11715) (isolated from soil samples from the Jammu hills) and characterized. The structure of the new compound 3 was established from 2D-NMR data. Chrysomycins A (1) and B (2) were identified using a strategic HPLC–PDA/LCMS and Dictionary of Natural Products (DNP) based fast dereplication. Additionally, two new naphthocoumarins, chrysomycins D and E were identified using LCMS, UV and DNP information. Chrysomycins A–C (1–3) were isolated for the first time from Streptomyces sporoverrucosus and were screened for cytotoxicity against a panel of cancer cell lines (A549, Colo205, PC-3, MIAPaCa-2, and HL-60), amongst which the most potent activity was observed against human leukemia HL-60 cells with IC50 values of 0.9, 0.95 and 11 μM, respectively. The mechanistic studies indicated that chrysomycins A (1) and B (2), at 1 μM concentration, distorted the cellular and nuclear morphology with significant DNA damage and apoptosis in HL-60 cells.