Microalgal biofilm growth on porous substrates has recently garnered extensive attention with the advantage of high biomass productivity with low liquid culture volume. In such cultivation, the surface characteristics of supporting materials play essential role supporting biofilm growth. In view of this, a study on the membrane surface wettability of a commercial polyethersulfone (PES) membrane with two different hydrophilicities was selected in a long-term benthic biofilm cultivation experiment. Findings revealed that both soluble and bounded extracellular polymeric substances (EPS) contributed towards the formation of an adhesive gel layer onto the membrane’s surfaces. The maximum accumulated cell density was achieved by C. fusiformis around 47.64 ± 4.03 × 109 cells m−2 onto phobic PES (79.29°) and the least by N. incerta around 6.40 ± 0.49 × 109 cells m−2 onto philic PES (47.56°). The fact remained that the cell attachment rate was almost similar for both A. coffeaeformis and N. incerta but with gradual reduction after day 12 for phobic PES and day 8 for philic PES for the three benthic species. The change of adhesion ratio was well explained with their respective EPS variation. The XDLVO model employed suggested the dominance of acid-base interactions in high ionic strength liquid, leading to higher global minimum energy occurring on phobic PES. Overall, the findings indicated that biofilm formation is favored on polymer substrate having lower surface energy due to the attractive interaction of the submerged surface with those biomolecules and colloids.