Polyhexamethyleneguanidine phosphate (PHMG-P) is a polymeric biocide with a guanidine group. It has multiple positive charges in physiological conditions due to nitrogen atom in the guanidine and this cationic property contributes antimicrobial effect by disrupting cell membranes. To determine whether the cationic nature of PHMG-P results in cytotoxicity in human cell lines, anionic compounds were treated with PHMG-P. The cytotoxic effect was evaluated with ROS production and HMGB1 release into media. To verify the protection effect of anion against PHMG-P-induced cell death in vivo, a zebrafish assay was adopted. In addition, membrane disruption by PHMG-P was evaluated using fluorescein diacetate and propidium iodine staining. As a result, anionic substances such as DNA and poly-L-glutamic acids, decreased PHMG-P induced cell death in a dose-dependent manner. While HMGB1 and ROS production increased with PHMG-P concentration, the addition of anionic compounds with PHMG-P reduced the ROS production and HMGB1 release. The mortality of the zebrafish increased with PHMG-P concentration and co-treatment of anionic compounds with PHMG-P decreased mortality in a dosedependent manner. In addition, FDA and PI staining confirmed that PHMG-P disrupts plasma membrane. Taken together, a cationic property is considered to be one of the main causes of PHMG-P-induced mammalian cell toxicity.