Membrane　fouling　has　been　a　challenge　for　the　practical　application　of　gravity-driven　membrane　(GDM)　systems.　In　this　paper,　gravity-driven　up-flow　slow　biofilter　(GUSB)　was　used　before　the　GDM　system　to　mitigate　membrane　fouling,　and　the　spatial　distribution　of　the　fouling　characteristics　was　investigated.　The　cake　layer　resistance　(Rc)　and　membrane　pore　resistance　(Rp)　were　reduced　after　GUSB　pretreatment,　resulting　in　a　62%　increase　in　permeate　flux.　In　the　membrane　module　vertical　direction,　Rc　and　Rp　declined　sequentially　from　top　to　bottom.　The　ratio　of　protein　to　polysaccharide　(PN/PS)　was　highly　negatively　correlated　with　nanoparticle　size　(Pearson＇s　r　=　−0.89),　and　was　the　key　factor　shaping　the　bacterial　community　(Mantel＇s　r　=　0.95).　The　increased　relative　abundance　of　functional　bacteria　(such　as　Candidatus_Obscuribacter　genus)　and　metabolic　functions　(amino　acid,　carbohydrate,　and　energy)　was　associated　with　the　structure　and　composition　of　the　biofouling　layer.　Rc　has　the　highest　weight　ratio　(0.699)　for　membrane　resistance.　Nanoparticle　size　influenced　surface　porosity　and　composition　of　the　biofouling　layer　by　regulating　bacterial　community　diversity　and　metabolic　functions,　which　indirectly　determined　membrane　resistance　(indirect　effect　coefficient　=　−2.018).　This　study　would　spark　a　wider　application　of　GDM　in　drinking　water　treatment,　and　provided　a　comprehensive　explanation　for　the　membrane　fouling　mechanism.　©　2023　Elsevier　Ltd