Preservatives in eye drops have always been the focus of people’s attention. Benzalkonium chloride (BAC) is one of the most frequently used bacteriostatic agents in eye drops, which has broad-spectrum and efficient bactericidal ability. However, the inappropriate dosage of BAC may lead to high cytotoxicity. Therefore, adding low-toxic hydroxypropyltrimethyl ammonium chloride chitosan (HACC) can not only achieve antimicrobial effect, but also have the advantages of moisturizing and biocompatibility. In this paper, the minimum inhibitory concentrations (MICs) of HACC and BAC were evaluated against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Diphtheroid bacillus and Candida albicans. Based on the MIC of each antimicrobial agent, an antimicrobial assay was performed to investigate the antimicrobial ability of disinfectant solution. Besides, cytotoxicity had also been assessed. When the HACC/BAC solution at weight ratio of 150/1 showed a highest antimicrobial efficiency and the cell proliferation rates were the highest in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Furthermore, the cell leakage was examined by UV absorption, indicating the great synergistic antimicrobial effect between HACC and BAC. What is more, the results of micromorphology research suggested that as the result of repulsive force between the two molecules, the average particle size of HACC would decrease. Finally, the impedance experiment showed that with the addition of BAC, current density would increase significantly, suggesting that more positive charge group was exposed to aqueous solution, leading the the increase of antimicrobial ability. Based on these results, HACC–BAC combination solution might be a promising novel antimicrobial group for biomedical applications.