Aspergillus fumigatus can lead to severe, life-threatening infections in individuals with compromised immune systems, pre-existing health conditions, or respiratory illnesses like COVID-19. Infections caused by azole-resistant A. fumigatus are particularly challenging to treat and have a high mortality rate. This research aimed to assess the effectiveness of eugenol encapsulated in chitosan biopolymer nanoparticles (NPs) in enhancing its antifungal properties against A. fumigatus. The study evaluated the physical characteristics of eugenol-encapsulated chitosan nanoparticles (EECNPs), including size, polydispersity, and eugenol release profile, using DLS, XRD, and FTRI techniques. The antifungal efficacy of the NPs against two A. fumigatus strains was tested through the CLSI-M38 microbroth dilution assay, killing time assay, reactive oxygen species (ROS) activity, and assessments of cell membrane integrity and cell wall morphology. The optimal eugenol/chitosan ratio was found to be 1.00:1.00, yielding the highest encapsulation efficiency (43.1%) and loading capacity (30.8%). The hydrodynamic radius of EECNPs was measured at 330.9 nm, with a polydispersity index (PDI) of 0.298 and a zeta potential of 18.9 mV. According to TEM micrographs, the average NP size was 300 nm. Antifungal activity was observed at MIC concentrations of 150-300 μg/mL. The killing time assay indicated that the nanoparticles could inhibit fungal growth after 12 hours, and toxicity tests showed over 60% survival of HEK293 cell lines at 2×MIC concentration. The release of reactive oxygen species (ROS) and extracellular potassium from A. fumigatus resulted in increased toxicity, damaging cell membrane integrity at a nanoparticle concentration of 600 µg/mL. Our findings demonstrated that encapsulating eugenol in chitosan biopolymer to create EECNPs not only extended the half-life of eugenol but also enhanced its antifungal activity against A. fumigatus through a combined mechanism that disrupts cell surfaces, maintains cell membrane integrity, and induces ROS activity as a serious fungal pathogen. This study is the first to report the antifungal mechanism of eugenol encapsulated in chitosan, highlighting its potential.