This study investigates the antibacterial activity of neat chitosan powder and flakes against three different bacterial species, Escherichia coli, Listeria innocua and Staphylococcus aureus, which are frequent causes of food spoilage. The effect of chitosan concentration and purity, as well as the influence of temperature, ionic strength (salt) and impact of a solid physical support in the medium are examined. Results show that the antibacterial activity of neat chitosan: (i) requires partial solubilisation; (ii) can be promoted by environmental factors such as adequate temperature range, ionic strength and the presence of a solid physical support that may facilitate the attachment of bacteria; (iii) depends on bacterial species, with a sensitivity order of E. coli > L. innocua > S. aureus; and (iv) increases with chitosan concentration, up to a critical point above which this effect decreases. The latter may be due to remaining proteins in chitosan acting as nutrients for bacteria therefore limiting its antibacterial activity. These results on the direct use of chitosan powder and flakes as potential antimicrobial agents for food protection at pH values lower than the chitosan pKa (6.2–6.7) are promising.