Nano-emulsion is a promising delivery system for increasing pesticide use and enhancing the therapeutic efficiency against pathogens. The pathogen Candidatus Liberibacter asiaticus (Las) that causes destructive citrus huanglongbing (HLB) resides in citrus phloem, which makes it difficult to treat with chemicals. Based on various physiochemical characteristics of oils, surfactants, and organic solvents, an oil-in-water (O/W) nano-emulsion formulation was developed and optimized to combat citrus HLB. The nano-emulsion was formulated through a spontaneous emulsification method for efficient delivery of ampicillin into the citrus phloem using bark application. The nano-emulsion that was prepared from Cremophor EL (viscous oil), acetone (water miscibility organic solvent), and Span 80/Tween 80 (surfactant) formed a small droplet size (17.33 ± 0.52 nm) and exhibited an improved absorption rate. Peak concentration was detected at 2 days posttreatment and the maximum concentration (Cmax) and relative bioavailability (RBA) of ampicillin in HLB-affected citrus were 71.86 ± 35.38 ng/g and 267.25% ± 44.1%, respectively. The peak concentration of Amp appeared at 6 days posttreatment in the citrus trees that were treated with Amp alone and their Cmax and RBA were 56.44 ± 32.59 ng/g and 100%, respectively. The same nano-emulsion was used to deliver five different antimicrobials to control citrus HLB through bark application. We found that the droplet size of the antimicrobials in the nano-emulsion was significantly reduced and the nano-emulsion also enhanced the therapeutic efficiency of validoxylamine A alone and in combination with actidione as well as sulfadimoethoxine sodium against Las. Therefore, this study provides an efficient bark application nano-emulsion formulation for citrus HLB control.