This work studies the antibacterial activity of chitosans prepared with controlled physicochemical characteristics against Gram-positive and Gram-negative reference bacteria. The optimal conditions in acetic acid and chitosan concentration were determined, and the disk diffusion method was used to evaluate the antimicrobial activity. All prepared chitosans, P CHTs, show an inhibition zone that varies between 14mm and 18mm depending on the strain and the physicochemical characteristics of the chitosan (degree of acetylation, DA; molar mass, Mv; and surface structure). These chitosans demonstrate enhanced antibacterial activity compared to the tested commercial chitosans, C CHTs. The effect of DA is particularly interesting. P CHTs exhibit significantly better antibacterial activity than C CHTs, especially against Gram-negative bacteria, where the improvement can reach 100%. Totally deacetylated chitosan (DA=0%) provided the highest inhibition diameter and the lowest MIC/MBC values. Also, PCHTs with Mv>80000 g/mol showed relatively high antimicrobial activity compared to Mv< 20000 g/mol. Interference of the surface structure with the effects of other parameters (Mv, DA, and strain) was observed. The antibacterial activity of the prepared chitosan (DA=0%) against E. faecalis was improved by 21% and 6% compared to Gentamicin and Ciprofloxacin. For the other bacteria, this chitosan provides 68 to 86% of the activity of Gentamicin and 49 to 80% of that of Ciprofloxacin.