The antimicrobial mechanism of naringenin, one of the citrus antibacterial flavonoids against food-borne Staphylococcus aureus ATCC 6538, was investigated in this study. Analysis of gas chromatography-mass spectrometry (GC-MS) and fluorescence showed that relatively low concentrations of naringenin caused perturbations in the membrane fatty acid composition and the conformation of membrane proteins through changing the microenvironment of phenylalanine, tyrosine, and tryptophan residues. Exposure of naringenin at higher levels significantly increased membrane permeability and changed the morphology of S. aureus cells. The genomic DNA-binding of naringenin was also quantitatively monitored using UV-vis spectra in combination with multivariate curve resolution-alternating least squares (MCR-ALS) analysis, and the concentration and pure spectra profiles for the three reaction species (DNA, naringenin, and DNA-naringenin) were obtained. Moreover, the thermal behavior of DNA and docking studies revealed that naringenin preferentially bound to the A-T base pair regions of genomic DNA via groove binding, and atomic force microscopy and circular dichroism showed that naringenin induced mild secondary structure and obvious morphological variations of this biomacromolecule. These results suggested that naringenin exerting its antibacterial effects might be connected with disruption of the cytoplasmic membrane and DNA targeting effects in Staphylococcus aureus.