Aims This study addresses the antibacterial activity and mechanism of action of BIOLL+(R), a commercial extract obtained from citrus fruits. Methods and Results Strong activities with minimum inhibitory concentrations (MIC) ranging from 10ppm (for some Brachyspira hyodysenteriae strains) to 80ppm (for various Salmonella enterica and Escherichia coli strains) were observed. Membrane integrity tests and Fourier transform infrared (FT-IR) spectroscopic analyses were performed to shed light on the effects caused on molecular structure and composition. Physical effects, with formation of pores and leakage of intracellular components, and chemical effects, which were dependent on the bacterial species, were evident on cellular envelopes. Whereas for S.enterica and E.coli, changes were focused on the carboxylic group of membrane fatty acids, for B.hyodysenteriae, the main effects were found in polysaccharides and carbohydrates of the cell wall. Conclusions The great antibacterial activity shown by BIOLL+(R) and its proposed dual physico-chemical mode of action, with species-specific cellular targets, show its attractiveness as an alternative to antibiotics. Significance and impact of the study Antibiotic resistance is becoming a serious problem. Our study characterizes a novel antimicrobial extract, which could represent an alternative to antibiotics for treatment or prevention of bacterial infectious diseases.