Fabrication of essential oil loaded-chitosan nanoparticles using electrospraying technique appears to be a novel strategy to develop thermally stable nanoparticles possessing higher encapsulation efficiency and particle stability. This study aims to fabricate chitosan nanoparticles (CNPs) loaded with Origanum vulgare essential oil (OEO, Origanum vulgare L.) at different proportions (OEO/CH proportions of 0:1, 0.0625:1, 0.125:1, 0.25:1 and 0.5:1 mL/g) using electrospraying technique. The CNPs were characterized in term of their particle size and stability (dynamic light scattering), encapsulation efficiency (spectrophotometry), and molecular (Fourier transform infrared spectroscopy), thermal (differential scanning calorimetry/thermogravimetric analysis), morphological (scanning electron microscopy) and antifungal (agar dilution method) and fungistatic activity properties. The average particle sizes of the CNPs ranged between 290 and 483 nm with a spherical morphology. Positively charged surface characteristics were observed to increase with the increment of OEO concentration in CNPs. The encapsulation efficiency values were determined in the range of 70.1 and 79.6%. The molecular and thermal analyses exposed very decent encapsulation of OEO into thermally stable chitosan nanoparticles. Morphological analysis verified the spherical shapes of these nanoparticles. Above all, the antifungal effectiveness of OEO against the Alternaria alternates AY1 could be significantly (p < 0.05) increased by its encapsulation into chitosan nanoparticles fabricated by the electrospraying technique. Consequently, it can be stated that the electrospraying technique developed is able to fabricate thermally stable nanoparticles owning higher encapsulation efficiency and particle stability. The results and findings suggest that the electrospraying technique would be a promising method to fabricate chitosan-based nanoparticles as an antimicrobial agent to control their release in a prolonged preservative effect in cosmetic, pharmaceutical and food applications for adjustable dosage forms.