Nanofiltration membrane fouling by oppositely charged polysaccharide (alginate) and protein (lysozyme) was systematically studied. It was found that membrane flux decline in the presence of both lysozyme and alginate was much more severe compared to that when there was only lysozyme or alginate in the feed solution. The flux performance for the mixed foulants was only weakly affected by solution pH and calcium concentration. These effects were likely due to the strong electrostatic attraction between the two oppositely charged foulants. Higher initial flux caused increased foulant deposition, more compact foulant layer, and more severe flux decline. The deposited foulant cake layer had a strong tendency to maintain a constant foulant composition that was independent of the membrane initial flux and only weakly dependent on the relative foulant concentration in feed solution. In contrast, solution chemistry (pH and [Ca²⁺]) had marked effect on the foulant layer composition, likely due to the resulting changes in the foulant-foulant interaction. The mixed alginate-lysozyme fouling could result in an initial enhancement in salt rejection. However, such initial enhancement was not observed when there was 1 mM calcium present in the feedwater, which may be attributed to the charge neutralization of the foulant layer.