Chitosan is a natural non-toxic, biocompatible, biodegradable, and mucoadhesive polymer. It also has a broad spectrum of applications such as agriculture, medical fields, cosmetics and food industries. In this investigation, chitosan nanoparticles were produced by an aqueous extract of Cympopogon citratus leaves as a reducing agent. According to the SEM and TEM micrographs, CNPs had a spherical shape, and size ranging from 8.08 to 12.01 nm. CNPs have a positively charged surface with a Zeta potential of + 26 mV. The crystalline feature of CNPs is determined by X-ray diffraction. There are many functional groups, including C꞊C, CH2-OH, C–O, C-S, N–H, CN, CH and OH were detected by FTIR analysis. As shown by the thermogravimetric study, CNPs have a high thermal stability. For the optimization of the green synthesis of CNPs, a Face centered central composite design (FCCCD) with 30 trials was used. The maximum yield of CNPs (13.99 mg CNPs/mL) was produced with chitosan concentration 1.5%, pH 4.5 at 40 °C, and incubation period of 30 min. The antifungal activity of CNPs was evaluated against phytopathogenic fungus; Fusarium culmorum. A 100% rate of mycelial growth inhibition was gained by the application of 20 mg CNPs/mL. The antitumor activity of the green synthesized CNPs was examined using 6 different cell lines, the viability of the cells reduced when the concentration of green synthesized CNPs increased, the IC50 dose of the green synthesized CNPs on the examined cell lines HePG-2, MCF-7, HCT-116, PC-3, Hela and WI-38 was 36.25 ± 2.3, 31.21 ± 2.2, 67.45 ± 3.5, 56.30 ± 3.3, 44.62 ± 2.6 and 74.90 ± 3.8; respectively.