The development of cleaning strategies for RO membranes processing highly charged wastewaters requires knowledge about foulant composition. The objective of this study was to characterize the fouling layer that formed on RO membranes processing swine wastewater pretreated by a mechanical solid-liquid separator (LF) and aerobic biofiltration (AE). The two LF effluents represented swine wastewater concentrated three (LF1) and four (LF2) times with BW30 (36 bar) and SW30 (60 bar) membranes, respectively. Flux decline was slightly higher with LF2-SW30 (37.1%) than LF1-BW30 (31.8%) after 20 h of filtration. The fouling layer had significantly higher protein concentration on LF2-SW30 than on LF1-BW30, but both had similar microbial content and inorganic composition. The AE effluents were concentrated by a factor of 7 and processed at pH 5.75 (AE1) and 8.07 (AE2) with BW30 membranes. Both AE effluents had higher calcium but lower suspended solids and organics than the LF effluents. They generated significantly higher flux decline and biofouling than LF1. High flux decline was also associated with increased protein attachment for AE1 and inorganic fouling for AE2. A mechanical separator may be adequate to prepare swine wastewater for RO filtration but the aerobic pretreatment would allow for a higher volumetric concentration of the effluent.