The characterization of membranes is suitable to investigate changes in the membrane properties caused by Layer-by-Layer (LbL) modification. Besides permeability, rejection, and molecular-weight cut-off (MWCO), which give information about the modification of the separation behaviour of the membrane, the zeta potential is capable of describing the surface charge of the membrane and its variation impacted by the properties of the polyelectrolyte multilayers (PEM). In this study, a new method for zeta potential measurement of hollow fibre membranes with several capillaries was developed and further investigations on the LbL modification of such membranes were performed. The results showed that an LbL coating with 8 DL PDADMAC/PSS led to a significant increase in the membrane charge of more than 20 mV. The coating with a different number of polyelectrolyte (PE) layers showed a zig-zag behaviour, comparable to data from flat sheet studies. However, in contrast to most flat sheet membranes, the charge curve assumes a totally negative trajectory at neutral pH. Further experiments on the MWCO of the LbL-modified membrane showed a reduction in the pore diameter from approx. 20 nm to less than 2 nm, reaching the range of nanofiltration membranes. With information on both the zeta potential and the MWCO, it was found that the rejection mechanism in LbL-modified multibore membranes is a complex interplay between the sieving effect due to reduction in the pore diameter and the repulsion effect of the charged membrane.