Chitosan, as a biocompatible polymer, is very attractive for biomedical applications. Continues studies are performing for improving its physicochemical features in order to make it more suitable for such approaches. In this study, methylated 4-N,N dimethyl aminobenzyl N,O carboxymethyl chitosan (MABCC) was synthesized,as a new chitosan derivative, in three steps. The investigations were carried out using FTIR, NMR, TGA and zeta potential measurement. Antibacterial and cell viability assessments were performed on four bacterial strains and two cell lines respectively. FTIR and NMR results showed that all substitution reactions were successfully carried out. Zeta potential of MABCC at various pH especially alkaline pH was greater than chitosan and it revealed increasing the solubility of the derivative. Antibacterial activity of MABCC was extremely greater than chitosan especially in Gram positive bacteria.Furthermore,it had no significant cytotoxicity against MCF-7 and Skov-3 cell lines in comparison to chitosan. These findings confirm that this new derivative can be introduced as a suitable compound for biomedical purposes.