Polyhexamethylene guanidine-phosphate (PHMG-P), a guanidine-based cationic antimicrobial polymer, is an effective antimicrobial biocide, potent even at low concentrations. Due to its resilient bactericidal properties, it has been used extensively in consumer products. It was safely used until its use in humidifiers led to a catastrophic event in South Korea. Epidemiological studies have linked the use of PHMG-P as a humidifier disinfectant to pulmonary fibrosis. However, little is known about its harmful impacts other than pulmonary fibrosis. Thus, we applied a zebrafish embryo/larvae model to evaluate developmental and cardiotoxic effects and transcriptome changes using RNA-sequencing. Zebrafish embryos were exposed to 0.1, 0.2, 0.3, 0.4, 0.5, 1, and 2 mg/L of PHMG-P from 3 h to 96 h post fertilization. 2 mg/L of PHMG-P resulted in total mortality and an LC50 value at 96 h was determined at 1.18 mg/L. Significant developmental changes were not observed but the heart rate of zebrafish larvae was significantly altered. In transcriptome analysis, immune and inflammatory responses were significantly affected similarly to those in epidemiological studies. Our qPCR analysis (Itgb1b, TNC, Arg1, Arg2, IL-1β, Serpine-1, and Ptgs2b) also confirmed this following a 96 h exposure to 0.4 mg/L of PHMG-P. Based on our results, PHMG-P might induce lethal and cardiotoxic effects in zebrafish, and crucial transcriptome changes were linked to immune and inflammatory response.