Size-controlled azoxystrobin-poly (lactic acid) microspheres (MS) were prepared by an oil/water emulsion solvent evaporation approach. The hydrated mean particle sizes of the MS1, MS2, and MS3 aqueous dispersions were 130.9 nm, 353.4 nm, and 3078.0 nm, respectively. The drug loading and encapsulation efficiency of the azoxystrobin microspheres had a positive relationship with particle size. However, the release rate and percentage of cumulative release were inversely related to particle size. The smaller-sized microspheres had a greater potential to access the target mitochondria. As a result, the more severe oxidative damage of Colletotrichum higginsianum Sacc and higher antagonistic activity were induced by the smaller particle size of azoxystrobin microspheres. The 50% lethal concentrations against Colletotrichum higginsianum Sacc of MS1, MS2, and MS3 were 2.0386 μg/mL, 12.7246 μg/mL, and 21.2905 μg/mL, respectively. These findings reveal that particle size is a critical factor in increasing the bioavailability of insoluble fungicide.