Herein, we illustrate the influence of citric acid (CA) concentration on the structural, morphological, optical, and antibacterial properties of the quaternary CuO-Fe2O3-MgO-CuFe2O4 nanocomposites (NCs). The target NCs were synthesized via auto-combustion method and characterized using XRD, FTIR, EDX, SEM, and UV-visible techniques. XRD analysis proved that CuO, Fe2O3, MgO, and CuFe2O4 were crystalized as monoclinic, hexagonal, cubic, and tetragonal, respectively. The energy gap, as a result of the combined effect of all the constituent substances, was decreased from 3.32 to 2.88 eV as the concentration of CA increased. Elemental analysis was further supported the NCs composition as, beside oxygen, Cu, Mn, and Fe metallic atoms. The antibacterial activity for the NCs, as assessed against Gram-negative (E. coli) and Gram-positive bacteria (S. aureus) was adequate, higher against S. aureus, and increased as CA concentration increased. Thus, by controlling the synthesis conditions like CA concentration, these quaternary NCs can be tailored to satisfy certain applications, allowing further research to be carried out.