Tea tree oil (Melaleuca alternifolia) has been acknowledged for its antibacterial and antifungal properties. Previous publications revealed that the tea tree oil has antibacterial activity against Propionibacterium acnes, which is the leading cause of acne, Staphylococcus aureus that caused various skin infections, and antifungal activity against Candida albicans. In this work, we aim to investigate the effect of microemulsion preparations in various gelling agents to their antibacterial and antifungal properties.Microemulsions have smaller droplets size than conventional emulsions; thus, we hypothesized that there would be a different effect on their inhibitory properties. The tea tree oil is formulated into a microemulsion and conventional emulsion, followed by incorporation into different gelling agents: Carbopol 940, Hydroxy-propyl methylcellulose, and sodium alginate. Physicochemical properties, such as pH, viscosity, and physical stability, had also been tested. The antibacterial and antifungal properties were tested using agar well diffusion methods. The microemulsion size was (26.23 + 0.15) nm, the pH of the final gel formulations was 4 to 7, and all formulations were physically stable under room temperature. The diameter of inhibition zones against bacteria and fungus was statistically analyzed using the multifactorial ANOVA method. The factors significantly affecting antibacterial and antifungal activities are the species of fungus and bacteria (p <0.001), gelling agents in formulations (p <0.05), and emulsions preparations (p <0.001). It was also interesting to note that these factors were significant on the activities tested (p <0.05).