N,N,N-trimethyl chitosan (TMC) with 93% degree of trimethylation was synthesized. TMC and the chitosan starting material were subjected to acidic hydrolysis to produce 49 different samples with a reduced average molecular weight (M_w) ranging from 2 to 144 kDa. This was done to allow the investigation of the relationship between antibacterial activity and M_w over a wide M_w range. NMR investigation showed that hydrolysis did not affect the degree of trimethylation (DS_TRI) or the structure of the polymer backbone. The activity of TMC against Staphylococcus aureus (S. aureus) increased sharply with M_w until a certain M_w value (critical M_w for high activity, CMW) was reached. After the CMW, the activity was not affected by a further increase in the M_w. A similar pattern of activity was observed for chitosan. The CMW was determined to be 20 kDa for TMC and 50 kDa for chitosan.