The objective of this study was to investigate the chemical composition of Syzygium aromaticum essential oil (SAEO), both as its toxicity and biological activities on Corynebacterium diphtheriae. The essential oil (EO) was obtained by hydrodistillation and verified by GC-MS. The main chemical components were eugenol (48.79%), caryophyllene (44.29%) and α-humulene (4.87%). In vitro tests with human red blood cells, blood compatibility. SAEO showed high toxicity for Artemia salina and the bioassay on Tenebrio molitor larvae revealed an average survival rate of 80.00%. The antioxidant activity of SAEO was moderate (40.60%) using the phosphomolybdenum method. SAEO inhibited the growth of all C. diphtheriae strains tested at minimum inhibitory concentrations of 62.5-1000 µg mL-1 (strong to moderate). The minimum bactericidal concentration of SAEO was observed in two strains. Microorganisms cultivated in the presence of subinhibitory concentrations (subMIC) do not show morphological changes. The subMIC of SAEO inhibited in some strains, but in most cases, increased biofilm formation. In conclusion, the results of this study found that SAEO has an inhibitory effect against C. diphtheriae, representing an alternative antimicrobial therapy, although more in vivo biological studies, alone or in combination with occasional antimicrobials, should be carried out to better evaluate its effects.