Fluorescence quenching dynamics were studied in poly(9,9-dioctylfluorene) films with a wide range of β-phase concentrations. Measurements of time-resolved fluorescence and fluorescence quantum yield showed that the radiative decay rate was independent of the β phase content, whereas the nonradiative decay rate was found to be higher in β-phase rich films. The trend was analyzed in terms of diffusion-mediated excitation energy transfer to quenchers. The concentration of fluorescence quenchers is found to be less than 0.01% of the number of fluorene repeat units and independent of the fraction of the film in the β phase. The results suggest that fluorescence quenching is predominantly by chemical defects rather than by excimer formation.