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Plant-associated fungal endophytes hold great potential to synthesize metabolites similar to their host, with potential anticancer effects. The present study aims to identify lead compound in the ethyl acetate (EA) extract of fungal endophyte, Penicillium oxalicum, associated with a medicinal plant Amoora rohituka, responsible for the anti-breast cancer activity. The crude extract of P. oxalicum was subjected to High-performance liquid chromatography (HPLC) analysis and further, bioactivity-guided fractionation was performed for HPLC fractions based on cell viability assay. The potential fraction further showed dose-dependent generation of nitric oxide in breast cancer cells that indicates their activity in the induction of oxidative stress. The characterization of potential fraction was done using different analytical techniques, such as liquid chromatography coupled with quadrupole time of flight mass spectrometry (LC-Q-TOF-MS), high-resolution mass spectrometry (HRMS), Fourier transform-infrared (FTIR) spectroscopy, and both ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy that validates the mass, structure, and functional groups of the lead compound, identified as Phytosphingosine. Further, DAPI assay revealed Phytosphingosine induced alteration of nuclear morphology in breast cancer cells, indicating apoptotic events. The blood compatibility of Phytosphingosine was evidenced through hemolysis assay, thus suggesting its safety profile. This is the first report of the purification and characterization of Phytosphingosine from P. oxalicum, exhibiting anti-breast cancer activity. The cytotoxic potential of Phytosphingosine against MDA-MB-231 and MCF-7 cells suggests further exploration to elucidate its molecular cross-talks and signalling pathways to develop targeted therapy against BC.