The objective of the present study was to prepare CuFe2O4 ferrite nanoparticles using the sol-gel combustion method, employing lemon juice as a surfactant and energy agent. This method is located within the green chemistry, representing an environmentally friendly and less expensive approach compared to other methods. The nanoparticles were subsequently evaluated as antibacterial agents against different pathogenic bacteria. Before the antibacterial assays, a cytotoxicity test was conducted to evaluate their safety when applied to organisms. The structural, morphological, elemental composition, and magnetic properties of the samples were analyzed using Fourier-Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD), Field Emission-Scanning Electron Microscopy (FE-SEM), and Energy Dispersive X-Ray Detection (EDX). The X-ray diffraction patterns confirmed both the phase purity and the particle size to be 24.27 nm. The results demonstrated that the CuFe2O4 nanoparticles exhibited substantial antibacterial activity against both Gram-negative bacteria (Sphingomonas paucimobilis) and Gram-positive bacteria (Staphylococcus lentus and Bacillus subtilis). The antibacterial efficacy was more pronounced against Gram-negative bacteria, with inhibition diameter 5.46mm and 10.64mm at concentrations of 5000 ppm and 10000 ppm, respectively. When making a comparison, the effectiveness against Gram-positive bacteria displayed a slight reduction. Inhibition zones measured 2.76 mm and 8.33 mm for Staphylococcus lentus, while they were 3.58 mm and 5.35 mm for Bacillus subtilis. These measurements were observed at nanoparticle concentrations of 5000 ppm and 10000 ppm, respectively. Furthermore, the study confirmed the safety of the CuFe2O4 nanoparticles by assessing their toxicity on human red blood cell at different concentrations (50, 100,250,500,1000,5000, and 10000 ppm).