This study explored how plantaricin Q7 acts against Pseudomonas fluorescens at cellular and protein expression levels. Plantaricin Q7 exhibited a minimum inhibitory concentration (MIC) of 144 AU/mL against P. fluorescens. The scanning electron microscopy and transmission electron microscopy revealed morphological changes in P. fluorescens upon exposure to 1 MIC of plantaricin Q7, disrupting cell membrane permeability and integrity, forming pores, and causing intracellular material leakage. The confocal laser scanning microscopy and leakage tests confirmed these observations. The attenuated total reflectance-Fourier transform infrared spectroscopy indicated alterations in cell proteins and carbohydrates due to plantaricin Q7. Proteomics analysis of bacterial protein content showed significant differences between untreated and plantaricin Q7-treated P. fluorescens, as confirmed by principal component analysis. Downregulation of MlaC, MlaF, FadA, FadB, PyrB, IlvC, GlnA, L29, L31, and L32 at protein and transcriptional levels post-treatment with plantaricin Q7 suggested inhibition of the maintenance of lipid asymmetry system primarily responsible for membrane damage. Furthermore, plantaricin Q7 inhibited fatty acid degradation, disrupted amino acid metabolism, and potentially suppressed ribosomal protein expression, revealing a new target for its anti-P. fluorescens activity. This study provides important insights into plantaricin Q7’s mechanism against P. fluorescens.