Fouling development in direct contact membrane distillation (DCMD) for seawater desalination was evaluated combining in-situ monitoring performed using optical coherence tomography (OCT) together with destructive techniques. The non-invasive monitoring with OCT provided a better understanding of the fouling mechanism by giving an appropriate sampling timing for the membrane autopsy. The on-line monitoring system allowed linking the flux trend with the structure of fouling deposited on the membrane surface. The water vapor flux trend was divided in three phases based on the deposition and formation of different foulants over time. The initial flux decline was due to the deposition of a 50–70 nm porous fouling layer consisting of a mixture of organic compounds and salts. Liquid chromatography with organic carbon detection (LC-OCD) analysis revealed the abundance of biopolymer in the fouling layer formed at the initial phase. In the second phase, formation of carbonate crystals on the membrane surface was observed but did not affect the flux significantly. In the last phase, the water vapor flux dropped to almost zero due to the deposition of a dense thick layer of sulfate crystals on the membrane surface.