Nanoencapsulation of Eucalyptus Essential Oils via Box–Behnken Design: Phytochemical Profiling and Enhanced Antibacterial and Antibiofilm Efficacy

The increasing burden of infectious diseases and antimicrobial resistance underscores the urgent need for alternatives to conventional therapeutics. Although <i>Eucalyptus</i> essential oils (EOs) are well recognized for their broad-spectrum antimicrobial and antibiofilm properties, their relatively high volatility and limited physicochemical stability restrict their practical applications. In this study, a systematic, design-driven comparative approach was employed to develop nanoemulsions containing <i>Eucalyptus globulus</i>, <i>E. citriodora</i>, and <i>E. radiata</i> EOs, with the aim of improving their stability and biological efficacy. Initially, gas chromatography-mass spectrometry was performed to identify species-specific chemotypes, which guided rational formulation design. A Box-Behnken design enabled the precise optimization of critical colloidal parameters, resulting in nanoemulsions with droplet sizes below 200 nm, polydispersity indices below 0.3, and ζ-potentials of approximately -22 mV. Comprehensive structural characterization by Fourier transform infrared spectroscopy and scanning electron microscopy, together with stress testing, confirmed the robust physical stability of the formulations. <i>In-vitro</i> antimicrobial evaluations against <i>Staphylococcus aureus</i>, <i>Enterococcus faecalis</i>, and <i>Klebsiella pneumoniae</i> revealed up to a 4-fold enhancement in antimicrobial activity and up to 80% inhibition of biofilm compared to the corresponding unformulated essential oils. The enhancements are most likely attributable to enhanced dispersion, increased interaction with greater microbial interfaces, and nanoscale stabilization of volatile bioactive constituents. Collectively, the optimized <i>Eucalyptus</i> nanoemulsions constitute reproducible, chemically well-defined nanocarrier systems with markedly improved antibacterial and antibiofilm performance, highlighting their promise as next-generation delivery systems for phytopharmaceutical applications. Further comprehensive biological and toxicological investigations are required to support the safe and effective translation of essential oil-containing nanocapsulations into practical use.