Spontaneous natural biofilm concentrates microalgal biomass on solid supports. However, the biofilm is frequently susceptible to exfoliation upon nutrient deficiency, particularly found in aged biofilm. Therefore, this study highlights a novel biofilm cultivation technique by pre-depositing the algal organic matters from marine diatom,
Navicula incerta
onto microporous polyvinylidene fluoride membrane to further strengthen the biofilm developed. Due to the improvement in membrane surface roughness and hydrophobicity, cells adhered most abundantly to soluble extrapolymeric substances-coated (sEPS) (76
10
6
16
10
6
cells m
−2
), followed by bounded EPS-coated (57.67
10
6
0.33
10
6
cells m
−2
), internally organic matter (IOM)-coated (39.00
10
6
5.19
10
6
cells m
−2
), and pristine control the least (6.22
10
6
0.77
10
6
cells m
−2
) at 24
th
h. Surprisingly, only bEPS-coated membrane demonstrated an increase in cell adhesion toward the end of the experiment at 72 h. The application of the bio-coating has successfully increased the rate of cell attachment by at least 45.3% upon inoculation and achieved as high as 89.9% faster attachment at 72 hours compared to the pristine control group. Soluble polysaccharides and proteins might be carried along by the cells adhering onto membranes hence resulting in a built up of EPS hydrophobicity (>70% in average on bio-coated membranes) over time as compared with pristine (control) that only recorded an average of approximately 50% hydrophobicity. Interestingly, cells grown on bio-coated membranes accumulated more internally bounded polysaccharides, though bio-coating had no discernible impact on the production of both externally and internally bounded protein. The collective findings of this study reveal the physiological alterations of microalgal biofilms cultured on bio-coated membranes.