In the present work, a convenient and direct technique which enables to characterize the intrinsic structure and the mechanical properties of the biofilm without altering its chemical and physical properties is proposed. By utilizing the Optical Coherence Tomography (OCT) as a structural imaging tool coupled with an advance mathematical framework, thickness, micro-porosity, normal stress-strain curve, bulk modulus and total permeability of the biofilm structures are determined. The accuracy of this mathematical technique for the in situ characterization is validated by analyzing two different membrane structures for porosity and permeability values against the mercury intrusion porosimetry method. Three-dimensional images of biofouling were obtained with high resolution aided to numerically analyze the intrinsic biofilm structure at microscale. Growth of biofilm in a dead-end filtration experimental setup was investigated by varying the feed flow rate which allowed uniform compression and decompression to compute normal stress-strain relation of the evolving biofilm structure. At early development of biofilm (day 3), the thickness and normal stress/strain curve showed that the biofilm structure behave similar to elastic material. However, hysteresis-like trend starts to appear with the growth of biofilm suggesting the deviation of biofilm properties to viscoelastic nature at day 8. The microstructure porosity increased from 0.214 (day 3) to 0.482 (day 8) at a feed flow rate of 15 mL/min. The total membrane/biofilm permeability decreased with biofilm age to reach 5.19 × 10−15 m2 at day 8 at the same flow rate, leading to a reduction of permeate flux over time. All the structural properties were found to be time dependent as the biofilm continuously evolved.