Hydrophobic acid organic matter (HpoA) extracted from treated effluent has been known to improve the rejection of steroid hormone estrone by reverse osmosis (RO) and nanofiltration (NF) membranes. In this study, the effects of solution chemistry (solution pH and ionic strength) on the estrone rejection by NF membrane with the presence of HpoA were systematically investigated. Crossflow nanofiltration experiments show that the presence of HpoA significantly improved estrone rejection at all pH and ionic strength levels investigated. It is consistently shown that the “enhancement effect” of HpoA on estrone rejection at neutral and alkaline pH is attributed to the binding of estrone by HpoA macromolecules via hydrogen bonding between phenolic functional groups in feed solutions, which leads to an increase in molecular weight and appearance of negative charge. The membrane exhibited the best performance in terms of estrone rejection at pH 10.4 (compared to pH 4 and pH 7) as a result of strengthening the electrostatic repulsion between estrone and membrane with the presence of HpoA. At neutral pH level, the ability of HpoA macromolecules to promote estrone rejection became stronger with increasing ionic strength due to their more extended conformation, which created more chances for the association between estrone and HpoA. The important conclusion of this study is that increasing solution pH and salinity can greatly intensify the “enhancement effect” of HpoA. These results can be important for NF application in direct/indirect potable water reuse.