<p>The objective of this study is to investigate the optimal ultrasonic power for producing microemulsions with <i>Ocimum gratissimum</i> L. essential oil and to evaluate their physicochemical properties. The effects of three powers (125&#xa0;W, 250&#xa0;W, 375&#xa0;W) on chitosan-based <i>Ocimum gratissimum</i> L. essential oil emulsion were examined by multi-frequency ultrasound-assisted homogenization. The results indicated that the physicochemical properties of chitosan-based <i>Ocimum gratissimum</i> L. essential oil emulsion were optimized under ultrasonic-assisted homogenization of three frequencies (20, 28, and 40&#xa0;kHz combined) at 125&#xa0;W for 15&#xa0;min. Compared to the control group, significant reductions in particle size were observed in the emulsion treated with 125&#xa0;W multi-frequency ultrasound (average particle size was 1.87 ± 0.01&#xa0;μm; zeta potential was 13 ± 0.53 mV). Furthermore, the stability and antioxidant activity of the emulsion showed marked improvements (the scavenging rates of DPPH and ABTS increased to 87.57% and 81.71%, respectively). In addition, scanning electron microscopy and confocal laser scanning microscopy images revealed that 125&#xa0;W multi-frequency ultrasonic-assisted homogenization emulsion droplets were evenly distributed and exhibited the highest encapsulation effect. These findings provide valuable insights into developing food-grade chitosan-based emulsions containing <i>Ocimum gratissimum</i> L. essential oil for functional food production.</p> Graphical abstract <p></p>