MAGNETIC nanocomposites loaded with antimicrobial drugs have grown in popularity for treating infectious diseases due to their magnetic properties and the ability to penetrate bacteria cells. In this research, CuxFe3-xO4 nanoparticles and CuxFe3-xO4/PANI nanocomposites were synthesized, characterized, and examined for their antimicrobial activity. The in-situ oxidative polymerization of aniline in the presence of CuxFe3-xO4 nanoparticles produced CuxFe3-xO4/PANI nanocomposites with distinct spinel ferrites. XRD, FT-IR, VSM, SEM, EDX, TEM, and XPS techniques confirmed the successful synthesis of the CuxFe3-xO4/PANI nanocomposites. The analysis of magnetization patterns of CuxFe3-xO4/PANI nanocomposites revealed their superparamagnetic characteristics. With increasing Cu2+ ratios, both the pure copper ferrites nanoparticles and the CuxFe3-xO4/PANI nanocomposites exhibited enhanced antimicrobial effects against gram-positive (Staph), gram-negative (E. coli, P. aeruginosa, and K. pneumoniae), and fungi such as (C. albicans), which is consistent with decreasing the order of crystal size (nm) and the lattice strain, where crystal size and lattice strain decreased with increasing Cu2+ ratio. SEM micrographs revealed changes in bacterial shape, deformation of fungal cell walls, and bacterial and yeast colonies collapsing. Furthermore, the nanocomposites are more effective against gram-negative bacteria and Candida albicans than gram-positive bacteria, displaying the alteration of bacterial shape, fungal cell wall deformation, and collapsing bacterial and yeast colonies. The results presented an approach for using CuxFe3-xO4 and CuxFe3-xO4/PANI as an alternative promising antimicrobial agent against several multidrug-resistant microbes.