There is a growing demand for chitosan production from fungi as it provides superior physico-chemical properties over traditional crustacean sources. Chitosan was recovered from Aspergillus niger biomass using different conditions of alkali deproteinization and Box-Benhken experimental design was evaluated to study the influence of alkali concentration, temperature and time on chitosan yield, degree of deacetylation (DD) and molecular weight (MW). Chitosan with different physico-chemical properties was obtained under different combinations of extraction factors. Desirability function was then used to predict the best overall combinations of responses. The most desirable compromise allowed for the recovery of chitosan with a yield of 7.0% (w/w), DD 83.64% and MW 2.70 × 104 Da, which were in good agreement with the predicted values. The recovered chitosan exhibited a good hydroxyl radical scavenging activity and ferrous ions chelating ability reached to 82.40% and 87.23%, respectively. Additionally, it demonstrated good emulsion stabilizing capacities with different vegetable oils. The results suggest that fungal mycelia can be used as a promising source of chitosan with important physico-chemical properties suitable for several applications such as food, pharmaceutical, and cosmetic industries.