To improve the hydrophilicity and antibacterial activity of eugenol, infinitely dilutive microemulsions of eugenol were constructed with Tween as the surfactant and lactic acid as the co-surfactant. The antibacterial activities of eugenol before and after micro-emulsification against Escherichia coli and Staphylococcus aureus were also investigated. The results of the pseudo-ternary phase diagrams showed that compared with Tween-40 and Tween-60, Tween-80 was more effective to increase the area of microemulsion single phase. The solubilization effect of lactic acid was improved with the increase lactic acid concentrations. When the mass ratio of eugenol and lactic acid was 1∶3, the area of eugenol microemulsion single phase was expanded to 78.8%. In the antibacterial activity test, the lower the Tween-80 content, the higher the lactic acid content in a certain scope, and the higher the antibacterial activity of eugenol microemulsions. When eugenol, lactic acid and Tween-80 was at a mass ratio of 1∶3∶6 in the microemulsion, the growth of two tested bacteria was completely inhibited at eugenol with a mass fraction of 0.05%. Moreover, the antibacterial activity of the complex system was significantly higher than that of single eugenol microemulsion without lactic acid. Therefore, microemulsions developed in this study could provide a feasible formulation for enhancing solubility and antibacterial effect of eugenol, and were expected to be used in the field of food antisepsis and preservation.