Conjugated polymer nanoparticles with incorporated antifade agents were prepared, and ensemble and single particle measurements showed that incorporation of antifade agents effectively improves the fluorescence quantum yield and photostability of the conjugated polymer nanoparticles, likely by a combination of triplet quenching and suppression of processes involved in photogeneration of hole polarons (cations), which act as fluorescence quenchers. The photostability of conjugated polymer nanoparticles and CdSe quantum dots was compared, at both the ensemble and single particle level. The results provide confirmation of the hypothesis that quenching by photogenerated hole polarons is a key factor limiting the fluorescence quantum yield and maximum emission rate in conjugated polymer nanoparticles. Additionally, the results indicate the involvement of oxygen in photogeneration of hole polarons. The results also provide insight into the origin of quenching processes that could limit the performance of conjugated polymer devices.