Recent findings regarding nanofiltration (NF) have led to indications that it can be successfully used for separation of various biological solutions. As a novelty, this paper is the first to investigate the impact of the feed pretreatment process on the NF membrane performance used for separation of 1,3-propanediol (1,3-PD) fermentation broths. For this purpose, prior to the NF process, the feed was purified by microfiltration (MF) and ultrafiltration (UF) processes. Subsequently, the long-term NF process was carried out with the use of a commercial, flat-sheet, thin-film, polyamide NF270 nanofiltration membrane. Thereinto, to determine the dominant fouling mechanism, Hermia’s model was used. With regards to the pretreatment processes performed, it has been determined that the MF membrane (0.14 µm) provided the reduction in the number of bacteria cells present in the permeate, while the UF membrane (450 Da) allowed obtaining the sterile permeate. Consequently, the NF permeate flux for the UF permeate was significantly higher. Analysis of the fouling mechanisms showed that during the separation of the MF permeate, formation of a cake layer on the NF membrane surface was dominant. In turn, with regards to the UF permeate, membrane blocking occurred in two separate phases involving standard blocking and then cake layer formation. Finally, a strategy of NF membrane cleaning with the use of sodium hydroxide (NaOH) solution has been proposed.