Factors associated with membrane organic fouling during secondary effluent organic matter (EfOM) microfiltration were explored. Ozone application and its effects on EfOM characteristics along with pre-ozonation effects on EfOM membrane fouling were also studied. Microfiltration tests with EfOM and its hydrophilic and hydrophobic fractions show that hydrophilic substances (HIS) have a stronger fouling potential. This is explained through analysis that reveals HIS possessing a higher macromolecular and carboxylic group content along with higher soluble microbial products (SMP) or SMP-like substances. Adhesion forces directly measured with atomic force spectroscopy (AFM) proved that HIS had the highest affinity to pristine and EfOM fouled membrane. Pre-ozonation effects include an increase in carboxylic and phenolic groups which may aggravate membrane fouling. However, the reduction of dissolved organic carbon (DOC) (including mineralization of small organic molecules) and breaking of large molecules are the dominant criteria for fouling reduction. (C) 2010 Elsevier B.V. All rights reserved.