In this study, the essential oil of Origanum vulgare was evaluated for putative antibacterial activity against six clinical strains and five reference strains of Aggregatibacter actinomycetemcomitans, in comparison with some antimicrobials. The chemical composition of the essential oil was analyzed, using chromatography (CG) and gas chromatography–mass spectrometry coupled (CG–MS). The major compounds in the oil were Carvacrol (32.36%), α-terpineol (16.70%), p-cymene (16.24%), and Thymol (12.05%). The antimicrobial activity was determined by an agar well diffusion test. A broth microdilution method was used to study the minimal inhibitory concentration (MIC). The minimal bactericidal concentration (MBC) was also determined. The cytotoxicity of the essential oil (IC50) was <125 µg/mL for THP-1 cells, which was high in comparison with different MIC values for the A. actinomycetemcomitans strains. O. vulgare essential oil did not interfere with the neutralizing capacity of Psidium guajava against the A. actinomycetemcomitans leukotoxin. In addition, it was shown that the O. vulgare EO had an antibacterial effect against A. actinomycetemcomitans on a similar level as some tested antimicrobials. In view of these findings, we suggest that O.vulgare EO may be used as an adjuvant for prevention and treatment of periodontal diseases associated to A. actinomycetemcomitans. In addition, it can be used together with the previously tested leukotoxin neutralizing Psidium guajava.