This study investigates the fluorescence quenching of a poly(p)-phenyleneethynylene) (1) based polyelectrolyte by positively charged and neutral macromolecules. This work shows that the change in the fluorescence yield of 1 depends on a number of factors, including electrostatic, hydrophobic, and energy transfer interactions with the quencher and also changes in the solution conditions such as concentration and ionic strength. The fluorescence quenching is attributed to the formation of aggregates that form upon addition of different quenchers to a solution of 1 and/or the solution conditions. The extent of 1's aggregation is shown to depend on the type of interaction between the polymer and the quencher, the concentration of the polymer, and the ionic strength of the solution.