Abstract The antimicrobial action of different components present in essential oils including carvacrol, cinnamaldehyde, thymol, squalene, rosmarinic acid, tyrosol, eugenol and β -Caryophyllene against Gram-positive and Gram-negative bacteria is here reported. Planktonic bacteria as well as a model of biofilm forming bacteria were challenged against those components being carvacrol, cinnamaldehyde and thymol the components with the highest antimicrobial action in both different settings. The potential synergy of some of those components against pathogenic bacteria was also analyzed. The antimicrobial mechanism of the different components was analyzed by means of flow cytometry and by electronic and confocal microscopy. Finally, subcytotoxic doses against mammalian cell lines are here reported to highlight the reduced cytotoxicity of those components against eukaryotic cells. Carvacrol, cinnamaldehyde and thymol showed the highest antimicrobial action of all the natural origin compounds tested and lower cytotoxicity against eukaryotic cells than conventional antiseptics such as chlorhexidine. The high inhibition in biofilm forming activity of those components highlight also their demonstrate benefits in reducing pathogenic microorganisms. Importance The use and misuse of antibiotics has led to the emergence of antibiotic resistance to human and animal pathogens. Compounds from natural sources such as animals, plants, and microorganisms have been proposed as renewed potential antimicrobial alternatives. The comparative antimicrobial action of different components commonly present in essential oils including carvacrol, cinnamaldehyde, thymol, squalene, rosmarinic acid, tyrosol, eugenol and β -Caryophyllene against S. aureus and E. coli is here reported. Carvacrol, cinnamaldehyde and thymol are the components with the highest antimicrobial action. Bacteria membrane disruption represents the bactericidal mechanism attributable to these compounds. In addition, the presence of carvacrol, cinnamaldehyde and thymol hinders S. aureus biofilm formation and partially eliminates preformed biofilms. Those components are less toxic to human cells than chlorhexidine.