Current treatments against bacterial infections have severe limitations, mainly due to the emergence of resistance to conventional antibiotics. In the specific case of Pseudomonas aeruginosa strains, they have shown a number of resistance mechanisms to counter most antibiotics. Human secretory RNases from the RNase A superfamily are proteins involved in a wide variety of biological functions, including antimicrobial activity. The objective of this work was to explore the intracellular antimicrobial action of an RNase 3/1 hybrid protein that combines RNase 1 high catalytic and RNase 3 bactericidal activities. To achieve this, we immobilized the RNase 3/1 hybrid on Polyetheramine (PEA)-modified magnetite nanoparticles (MNPs). The obtained nanobioconjugates were tested in macrophage-derived THP-1 cells infected with Pseudomonas aeruginosa PAO1. The obtained results show high antimicrobial activity of the functionalized hybrid protein (MNP-RNase 3/1) against the intracellular growth of P. aeruginosa of the functionalized hybrid protein. Moreover, the immobilization of RNase 3/1 enhances its antimicrobial and cell-penetrating activities without generating any significant cell damage. Considering the observed antibacterial activity, the immobilization of the RNase A superfamily and derived proteins represents an innovative approach for the development of new strategies using nanoparticles to deliver antimicrobials that counteract P. aeruginosa intracellular infection.