Aspergillus fumigatus can cause an invasive life-threatening infection in people who have weakened immune systems, underlying diseases or respiratory infections such as COVID-19. Azole-resistant A. fumigatus infections are difficult to treat and have high mortality. This study aimed to reveal the efficacy of encapsulated eugenol in chitosan biopolymer nanoparticles (NPs) to increase its antifungal activity against A. fumigatus. The physical properties of eugenol-encapsulated chitosan nanoparticles (EECNPs), size, polydispersity and the release profile of eugenol were examined by DLS, XRD and FTRI techniques. The antifungal activity of the NPs against two strains of A. fumigatus were examined using CLSI-M38 microbroth dilution assay, killing time assay, reactive oxygen species (ROS) activity, destruction of cell membrane integrity/permeability, and cell wall morphological damage. The eugenol/chitosan ratio of 1.00:1.00 was considered the optimal range with the highest encapsulation efficiency (43.1%) and loading capacity (30.8%). The hydrodynamic radius of EECNPs was 330.9 nm, with polydispersity index (PDI) of 0.298 and the zeta potential of 18.9 mv. The average size of the NPs was 300 nm according to TEM micrographs. The antifungal activities were determined at 150-300 μg/mL as MIC concentration. The killing time assay showed that the nanoparticles could inhibit the fungal growth after 12 hours and toxicity revealed more than 60% survival of HEK293 cell lines in 2×MIC concentration. The release of reactive oxygen species (ROS), and extracellular potassium in A. fumigatus induce more toxic effects on damaging cell membrane integrity, through increasing 600 µg/mL concentration of nanoparticles. Our results revealed that the encapsulation of eugenol in chitosan biopolymer to produce EECNPs was not only increased the half-life of eugenol but also improved its antifungal activity against A. fumigatus by a combined mechanism of damaging cell surfaces, cell membrane integrity, and the induction of ROS activity as a life-threatening fungal pathogen. This study is the first to report that the antifungal mechanism of the eugenol encapsulated in chitosan has the potency of natural antimicrobial agents due to altering the destructive properties in A. fumigatus.