Recently, the use of chemical and synthetic preservatives for the control of microbial pathogenesis growth has been remarkably decreased. Today, essential oils (EOs) have been considered to be natural preservatives. Fennel is among the aromatic medicinal plant that can be used as a natural preservative. In this study, to investigate the antimicrobial activity of fennel essential oil on some pathogenic microorganisms causing infection and food poisoning (Escherichia coli, Staphylococcus aureus and Candida albicans) were used disk diffusion agar (Kirby-Bauer), well diffusion agar, minimum inhibitory concentration and minimum bactericidal/fungicidal concentration methods. The interaction of fennel essential oil (FEO) with kanamycin antibiotic was investigated. The results disc diffusion agar and well diffusion agar antimicrobial assays showed that S. aureus was the most sensitive bacteria with the highest inhibition zone compared to the other microorganisms. The effects of interaction of FEO with kanamycin antibiotic on Escherichia coli, Staphylococcus aureus showed synergistic status. In general, FEO was able to prevent the growth of Escherichia coli, Staphylococcus aureus and Candida albicans “in vitro”.