AbstractCopper oxide nanoparticles (CuO‐NPs) were functionalized with specific antibodies to target their antibacterial activity against Gram‐positive or Gram‐negative bacteria. The CuO‐NPs were covalently functionalized to cover their surface with specific antibodies. The differently prepared CuO‐NPs were characterized by X‐ray diffraction, transmission electron microscopy and dynamic light scattering. The antibacterial activities of the unmodified CuO‐NPs and the antibody‐functionalized nanoparticles (CuO‐NP‐AbGram−and CuO‐NP‐AbGram+) were determined for both Gram‐negativeEscherichia coliand Gram‐positiveBacillus subtilisbacteria. The antibody‐functionalized NPs showed a differential increase of their antibacterial activity according to the specific antibody. The CuO‐NP‐AbGram−inE. colishowed reduced half maximal inhibitory concentration (IC50) and minimum inhibitory concentration (MIC) values when compared with unfunctionalized CuO‐NPs. On the other hand, the CuO‐NP‐AbGram+also showed reduced IC50and MIC values inB. subtilis, when compared with non‐functionalized CuO‐NPs. Thus, the functionalized CuO nanoparticles with specific antibodies showed enhanced specificity of their antibacterial activity. The advantages of “smart” antibiotic nanoparticles are discussed.