Anticancer agents from microbial sources are gaining importance based on their safety and also due to the broad range of novel metabolites produced by them. In the present study, anti-cancer potential of a pink coloured bioactive fraction B5, extracted from Bacillus endophyticus JUPR15 was assessed on the cancer cells HeLa, HepG2, MCF-7 and toxicity was tested on normal human lymphocytes. Treatment with B5 resulted in a significant reduction in the survival of the cancer cells with the IC50 value ranging from 6.5 to 13.2 mu g/ml. At the molecular level, the apoptotic cell death mediated by B5 was correlated with the upregulation of p53 and Bax genes along with the downregulation of Bcl-2 as per the results of flow cytometry and qRT-PCR. In response to the apoptosis-inducing stimulus of B5, activation of caspases along with cell cycle arrest at G1 phase were evidenced in the cancer cells. Non-toxic nature of B5 to normal human peripheral lymphocytes was also confirmed throughout the study. Characterization of the compound through GCMS method indicated the presence of 9-Octadecenoic acid as one of the major components along with a thiophene derivative and a dinitrobenzamide derivative.