The main focus of this study was to create a stable and efficient nanoemulsion (NE) using Callistemon citrinus essential oil (EO). Various factors affecting the NE’s stability were optimized including oil %, Tween 80%, time of sonication, and its accelerated stability was examined. The research also considered the antibacterial, antifungal, and larvicidal effects of the optimized NE (B10). The optimum NE stood out for its stability, featuring a particle size of 33.15 ± 0.32 nm. Analysis via IR spectroscopy confirmed successful EO encapsulation in B10. The formulation remained stable for six months, with B10 showing significantly higher antibacterial and antifungal potency compared to the pure oil. When samples were subjected to tests against Fusarium oxysporum, B10 exhibited a MIC value of 62.5 mg/mL, whereas the pure oil showed a MIC value of 250 mg/mL. This indicates that the B10 formulation was 50 times more effective than the EO. In terms of antibacterial activity against Escherichia coli, the MIC value was 0.256 mg/mL for B10 and 4 mg/mL for the EO. Also, pure oil and B10 displayed larvicidal effects against Chilo suppressalis (Walker) larvae, with B10 eliminating 95.2% of larvae in 48 h. Overall, stable and optimum C. citrinus NE with its strong antimicrobial qualities, shows promise as an effective fungicide and insecticide
