Chitosan is used as an antimicrobial agent in different agri-food applications; in winemaking, the use of chitosan from Aspergillus niger is authorized, but other sources of chitin, and consequently of chitosan, are available, such as crustaceans and insects. This work investigates the antimicrobial efficiency of chitosan from crustaceans and insects (Hermetia illucens) against non-Saccharomyces yeasts in wine. For this aim, the first step was to evaluate the effect of crustacean chitosan, tested both alone and in combination with low sulphur dioxide (SO2) concentrations, on the cell viability of 20 non-Saccharomyces strains in the first step of fermentations inoculated with each strain. Furthermore, the strain resistance to crustacean- and insect-based chitosan was evaluated in agarized media, together with the addition of different antimicrobial concentrations. Finally, the efficiency of different antimicrobial treatments was evaluated during laboratory-scale fermentations inoculated with a selected S. cerevisiae strain. The tested strains exhibited medium/high resistance to the chitosan; in some cases, the behaviour varied in the function of species/strain, and only four strains exhibited different resistance levels, depending on the chitosan source. The addition of chitosan alone during fermentation inoculated with S. cerevisiae showed lower antimicrobial activity than SO2, but the combined use with SO2 showed a better effect than chitosan alone. The evaluation of the suitability of chitosan obtained from a sustainable source, such as insects, will allow us to give new information on the future applications of this natural compound for the production of wine with low sulphite content.