This study presents a comparative account on synthesis and characterization of ZnO/CuO (ZC) nanostructures by exploiting the capping, stabilizing and reducing characteristics of the biogenic green surfactants (rhamnolipid and surfactin) vis-à-vis chemical surfactant (sodium dodecyl sulfate). The ZC material is characterized by various sophisticated analytical tools such as X-ray diffraction (XRD), spectroscopic, and microscopic methods. The optical studies reveal the bandgap energy of the pristine ZC to be 3.77 eV, which varies marginally for SDS@ZnO/CuO (3.65 eV), rhamnolipd@ZnO/CuO (3.69 eV) and surfactin@ZnO/CuO (3.75 eV). In addition, antibacterial activities of the prepared samples against two pathogenic bacteria, Micrococcus luteus (ATCC 4698) and Escherichia coli DH5α (CP026085) are investigated and the mode of action is demonstrated through the destabilization and disruption of the bacterial membranes and subsequent killing.