Reducing the consumption of synthetic insecticides and increasing the use of natural and safe products have become the new strategies for crop protection. This study aimed to evaluate molecular networking-based chemical components using the new molecular networking strategy based on Global Natural Products Social (GNPS) web platform and biological activities of Melaleuca leucadendra essential oil against red imported fire ants (Solenopsis invicta). Results indicated that various chemical classes of this essential oil were identified, including monoterpenes and their derivatives, monoterpene aldehydes, and phenolics. The M. leucadendra essential oil with LC₅₀ approximately 146.58 ± 3.75 mg/mL exhibited fumigant toxicity against worker ants and also showed a significant repellent effect on S. invicta during outdoor environmental tests. Based on the analysis results of chemical compositions and the toxic activity against S. invicta of M. leucadendra essential oil, we suggested that the major compound, Methyleugenol, significantly contributed to the insecticidal ability. These results could prompt further research about natural, safe, and non-polluting insecticides for the low-income populations of the tropics.