The application of ozone pre-treatment for ultrafiltration (UF) in drinking water treatment has been studied for more than 10 years, but its performance in mitigating or exacerbating membrane fouling has been inconclusive, and sometimes contradictory. To help explain this, our study considers the significance of the influent organic matter and its interaction with ozone on membrane fouling, using solutions of two representative types of extracellular polymeric substances (EPS), alginate and bovine serum albumin (BSA), and samples of surface water. The results show that at typical ozone doses there is no measurable mineralization of alginate and BSA, but substantial changes in their structure and an increase in the size of nano-particle aggregates (micro-flocculation). The impact of ozonation on membrane fouling, as indicated by the membrane flux, was markedly different for the two types of EPS and found to be related to the size of the nano-particle aggregates formed in comparison with the UF pore size. Thus, for BSA, ozonation created aggregate sizes similar to the UF pore size (100 k Dalton) which led to an increase in fouling. In contrast, ozonation of alginate created the nano-particle aggregates greater than the UF pore size, giving reduced membrane fouling/greater flux. For solutions containing a mixture of the two species of EPS the overall impact of ozonation on UF performance depends on the relative proportion of each, and the ozone dose, and the variable behaviour has been demonstrated by the surface water. These results provide new information about the role of nano-particle aggregate size in explaining the reported ambiguity over the benefits of applying ozone as pre-treatment for ultrafiltration.