Polyquaternium polymers are widely used in various applications, such as personal care products and wastewater treatment plants, and eventually end up in the aquatic environment. While polymers have been perceived of low toxicological concern due to their size, several studies have pointed towards water-soluble cationic polymers being toxic towards aquatic organisms – and that the toxicity largely is determined by the polymer charge density. The present study investigated the polyquaternium toxicological mechanism of action throughout lipidomic analysis and changes in immune-gene expression (qPCR) of zebrafish larvae exposed continuously to two water-soluble polymers; a high charge density polyquaternium-6 and a low charge density polyquaternium-10, for 5 and 12 days upon fertilization. The results showed that the investigated polyquaterniums cause both inflammatory responses and significant alterations of the zebrafish larvae lipidome. Depending on polyquaternium polymer and larvae development stage, the gene expression showed an inflammatory response (e.g. significant up-regulation of il8, il1β and tnfα) in the exposed zebrafish. Alterations of the lipidome were additionally observed, with severe depletion of lipids (e.g. lyso-glycerophosphocholines and ceramides) in the 12 days old larvae exposed to high charge density polymer. The findings furthermore support a hypothetical mechanism of action to be non-specific and lethality potentially to be narcosis-like driven.