Surface waters contain colloids and natural organic matter, largely composed of humic substances. In this work the effect of natural organic matter (NOM) and humic substances (IHSS stream humic and fulvic acid reference material) on the deposition and rejection of inorganic colloids (hematite) by GVWP and GVHP microfiltration (MF) membranes was studied. Parameters of interest were solution pH, ionic strength, calcium concentration, primary colloid size (75, 250 and 500 nm), organic-type and concentration, as well as membrane-type and hydrophobicity, aggregate structure and colloid stability. The method of preparation of the equilibrated suspensions, and thus their aggregation state, had a large influence on the rejection of colloids and their aggregates, as well as the association of particles with the membrane material and flux decline. The systems studied were grouped into (a) organics in the absence of inorganic colloids, (b) stable primary particles, (c) primary particles at pH extremes with organics, (d) particles pre-aggregated in electrolyte solution prior to adsorption of organics (SPO), and (e) particles stabilised with organics (OPS). Extreme pH conditions and pre-adsorption of organics onto the particle surface created very stable systems (colloids retained their primary particle size) and the deposition of the colloids at the membrane surface was reduced significantly. This led to a penetration of the particles into the pores and adsorption on the pore walls leading to full rejection in the absence of organics. Flux decline was, in this case, dependent on colloid size, with the size closest to the membrane pore size causing the greatest flux decline. In the presence of organics, membrane–colloid charge interaction and adsorption were reduced and rejection decreased to near-zero for these stable colloid systems. For aggregates the presence of organics led to a greater flux decline. Rejection of colloids was complete, now determined by the large size of the aggregates formed, which also indicates great mechanical stability of these aggregates. Calcium played a key role in the flux decline of all systems. In the absence of hematite, calcium contributed to organic aggregation and increased flux decline. Calcium led to an increased flux for hematite aggregates (SPO) presumably due to the formation of looser aggregates and a decreased flux for stable colloid–organic systems (OPS) due to destabilisation of these systems.