Surface heterogeneity plays an important role in controlling colloidal phenomena. This study investigated the self-aggregation and bacterial adsorption of self-assembled monolayer coated gold nanoparticles (AuNPs) with different surface compositional and structural heterogeneity. Evaluation was performed on AuNPs coated with (1) one ligand with charged terminals (MUS), (2) two homogeneously distributed ligands with respectively charged. and nonpolar terminals (brOT) and (3) two ligands with respectively charged and nonpolar terminals with stripe-like distribution (OT). The brOT particles have less negative electrophoretic mobility (EPM) values, smaller critical coagulation concentration (CCC) and larger adsorption rate on Escherichia coli than that of AuNPs with homogeneously charged groups, in good agreement with DLVO predictions. Although the ligand composition on the surface of AuNPs is the same, OT particles have less negative EPM values and faster rate of bacterial adsorption, but much larger CCC compared to brOT. The deviation of OT particles from brOT and MUS in their self-aggregation behavior reflects the effects of surface heterogeneity on electrical double layer structures at the interface. Results from the present study demonstrated that, besides chemical composition, organization of ligands on particle surface is important in determining their colloidal stability.