Membrane fouling needs to be mitigated for widespread use of membrane bioreactors (MBRs). It has been pointed out that particles with small sizes found in supernatants (sub-micron particles) of mixed liquor suspensions of MBRs are important in the evolution of membrane fouling of this technology. However, information on characteristics of sub-micron particles in MBRs is still insufficient. In this study, a pilot-scale MBR treating municipal wastewater was used to investigate and characterize sub-micron particles in an MBR and to identify the size fraction(s) responsible for irreversible fouling in an MBR. It was clearly shown that characteristics of sub-micron particles in the MBR varied considerably depending on their sizes. Results of Fourier transform infrared (FTIR) analysis and monosaccharide analysis suggested that irreversible fouling in this study was mainly caused by the specific size fraction of 0.1–0.45 μm, which was close to the size of micropores of the membrane used. Pore plugging might explain this to some extent.