The present study has been designed to fabricate fungal endophyte assisted gold nanoparticles and elucidate their anti-breast cancer potential. The aqueous extract of fungal endophyte, Penicillium oxalicum, associated with the medicinal plant Amoora rohituka has been used for the fabrication of gold nanoparticles (POAuNPs). The physicochemical characterization using UV-Vis spectroscopy, FTIR, XRD, DLS, Zeta potential, TEM and FESEM analysis revealed stable, uniform distribution, spherical-shape and crystalline nature of POAuNPs with size range of 3-46 nm. Further, POAuNPs potentially inhibited the growth of pathogenic bacterial strains, E. coli and S. aureus. The synthesized POAuNPs has shown potential antioxidant effects against DPPH, superoxide and nitric oxide radical scavenging assay with an EC50 value of 8.875±0.082, 52.593±2.506 and 43.717±1.449 µg/mL, respectively. Moreover, the value of EC50 for total antioxidant capacity of POAuNPs was found to be 23.667±1.361 µg/mL. The cell viability of human breast cancer cells, MDA-MB-231 and MCF-7 was found to be reduced after treatment with POAuNPs and IC50 values were found to be 19.753±0.640 and 35.035±0.439 µg/mL respectively. Further, in vitro biochemical assays revealed POAuNPs induced metabolic reprogramming in terms of reduced glucose uptake and increased LDH release and, disruption of oxidative balance through depletion of GSH level, increased nitric oxide level and lipid peroxidation as a possible pathway to suppress the human breast cancer cell proliferation. Apoptosis-specific nuclear modulations induced by POAuNPs in human breast cancer cells were validated through DAPI nuclear staining. The present investigation thus attempted to show first ever fabrication of gold nanoparticles using aqueous extract of P. oxalicum associated with A. rohituka. The results revealed unique physico-chemical characteristics of myogenic gold nanoparticles and screening their effect against breast cancer via metabolic reprogramming and induction of apoptosis thus adds great significance against cancer therapeutics, suggesting further exploration to develop nanotherapeutic drugs.