Nowadays, consumers are demanding for minimally processed, additive-free, high quality, fresh-like and microbiologically safe foods. However, as food deterioration is the constant threat along the entire food chain, food preservation remains as necessary today as in the past. In this paper, several citrus essential oils, including sweet orange, lemon and lime essential oil applied directly in fresh orange juice as a natural preservative, were analyzed for their antimicrobial activity. The antimicrobial activity of each citrus essential oil was assessed against Saccharomyces cerevisiae, Lactobacillus plantarum and Aspergillus niger using broth macrodilution. Among all citrus essential oils tested, sweet orange essential oil with 0.10% concentration showed significant antimicrobial activity against microorganisms tested. Hence, it was chosen to be applied in the fresh orange juice. Then, shelf life analysis was conducted during 7 d of storage at 5 °C and during 2 d of storage at 25 °C using selected concentration of the selected citrus essential oil in the fresh orange juice. Based on the results obtained, the addition of sweet orange essential oil with 0.10% concentration was not able to prolong the shelf life of the fresh orange juice according to the Indonesian National Standard (SNI). However, the formulation of fresh orange juice itself was able to improve its shelf life when compared to the shelf life of the commercial orange juice.