Natural environment is a wealthy source of bionanofactories that invested in green approaches as the fabrication of biomimetic nanomaterials. The current study points out the importance of microbial activity in metal bioremediation, green synthesis of NPs, and global biogeochemical cycles of bioactive metals as well. It describes for the first time the synchronous biosynthesis of zero- (intracellular) and one-dimensional (extracellular) copper oxide nanoparticles (CuO-NPs) viaProteus mirabilis10B. This bionanofactory represents key location of reduction and stabilization, and its exopolysaccharide additionally provides nucleation and growth site for CuO-NPs. The as-synthesized CuO-NPs were characterized; UV-Vis spectroscopy revealed surface plasmon resonance at 275 and 430 nm for intracellular and extracellular CuO-NPs, respectively. XRD reflected crystalline, pure phase monoclinic structure CuO-NPs. EDX illustrated strong copper signal with atomic percentages 32.3% (intracellular) and 14% (extracellular) CuO-NPs. However,ζ-potential recorded −62.5 and −43.8 mV with PDI 0.207 and 0.313 for intracellular and extracellular CuO-NPs, respectively, confirming the colloidal stability and monodispersity. Moreover, TEM micrographs depicted quasi-spherical intracellularly sequestered CuO-NPs (10 nm). Unexpectedly, extracellular CuO-NPs exhibited rod-, needle-, and wire-shaped with 17–37.5 nm in width and 112–615 nm in length. The antagonistic activity of both types of CuO-NPs was evaluated against Gram-negative and Gram-positive bacteria (aerobic and anaerobic), biofilm, yeast, mold, and algae. The potent antagonistic efficacy of CuO-NPs was displayed which encourages its utilization in controlling microbial contamination. Finally, the promising metabolic activity ofProteus mirabilis10B can be exploited in simultaneous and beneficial applications for human and the surrounding ecosystem.