The interactions of antimicrobial poly(phenylene ethynylene) (PPE)-based cationic conjugated polyelectrolytes (CPEs) with lipid membranes were investigated to gain insight into the mechanism of their biocidal activity. Three model membrane systems comprising negatively charged phosphatidylglycerol (PG) lipids were used to mimic the bacterial cell membrane, including unilamellar lipid vesicles in aqueous solution, lipid bilayer coated silica microspheres, and lipid monolayers at the air-water interface. Two PPE CPEs, one containing a thiophene moiety on the PPE repeat unit and the second containing a diazabicyclooctane (DABCO) moiety on the pendant side chain, were chosen, since the former exhibits distinct dark biocidal activity and the latter shows strong light-activated antimicrobial activity but little dark biocidal activity. The interactions of these two PPE polymers with lipid membranes were characterized in detail by CPE fluorescence spectral changes, fluorescence resonance energy transfer (FRET), fluorescence quenching, monolayer insertion, and dynamic light scattering assays. Both PPE polymers exhibit affinity for the anionic lipid membrane systems. Their concomitant association and insertion into the membrane leads to conformational changes of the PPE polymer from an aggregated state to a more extended state, as evidenced by the polymer's enhanced fluorescence and FRET between the polymer and rhodamine incorporated in the lipid membrane. In comparison, the thiophene polymer exhibits stronger interactions with PG lipid membranes than the DABCO-containing polymer. The former induces a larger fluorescence enhancement, shows faster transfer across the lipid membrane, and inserts more readily and to a higher extent into lipid monolayers. The observed differences between the two PPE polymers in their interactions with the lipid membrane may stem from their structural differences, as the DABCO-containing polymer has a much bulkier and larger pendant group on its side chain. The higher degree of membrane interaction and insertion, and subsequent membrane disorganization, of the thiophene polymer may account for its dark biocidal activity.