The aim of the present study was to identify cytotoxic secondary metabolite produced by marine Streptomyces paradoxus VITALK03 and to study its mechanism of anticancer activity. Bioactivity guided screening of secondary metabolites from S. paradoxus VITALK03 resulted in identification of (3R,8aR)-3-(2-methylpropyl)-octahydropyrrolo[1,2-a]piperazine-1,4-dione (gancidin W). The anticancer cytotoxic potential of gancidin W was screened by in silico docking analysis using 22 cancer drug target proteins. Gancidin W showed the least binding energy of -7.55 Kcal/mol with breast cancer protein KRas of pERK pathway, and showed a Ki value of 2.94 μM and formation of 4 H-bonds during the interaction. The results of in silico docking and molecular dynamic studies were further confirmed by in vitro and in vivo anticancer cytotoxicity studies. Gancidin W showed selective cytotoxicity on breast cancer cells (MCF-7) with an IC50 value 1.56 μg/mL, while value of the same was 12.5 μg/mL in case of VERO cells (control). Administration of gancidin W (3 μg/d) for 14 d to MCF-7 injected (grafted) zebra fish resulted in 50% reduction in tumour progression. This is the first cumulative (preclinical) report on in vitro, in vivo and in silico anticancer activity of gancidin W. The results of in vitro, in vivo, molecular docking and molecular dynamic stimulation studies suggest that gancidin W could be probed further to develop as a breast cancer drug.