Membrane distillation (MD) is a promising desalination technology which allows to achieve high salt rejection at low energy expenses as compared to conventional desalination processes. However, just like in any other membrane separation process, the MD membrane is susceptible to biofouling which is one of the critical problems in membrane-based systems. In this study, we investigated the effects of spacer design and feed temperature on the biofilm formation and proliferation in a flat-sheet direct contact membrane distillation (DCMD) used for desalination of the Red Sea water. Two types of spacers (Standard & 1-Hole) were designed to evaluate their efficiency in biofouling mitigation at three different feed water temperatures (47 °C, 55 °C and 65 °C). Our results showed that while 1-hole spacer was more efficient in reducing biofouling at 47 °C (permeate flux declines of 73.2% and 79.6% after 5 days of DCMD process using 1-hole and standard spacers, respectively). Standard spacer over-performed at higher feed water temperatures (65.7%, and 75.2% after 5 days of DCMD process at 55 °C and 65 °C, respectively). The Optical Coherence Tomography (OCT) revealed a significant transition of biofilm morphology with increasing feed water temperature for both types of spacers. While thicker and more porous biofouling structures were formed on the surface of MD membrane at 47 °C and 55 °C, thinner non-porous layer prevailed on the membrane surface at a feed water temperature of 65 °C. This observation was supported by direct enumeration of bacterial cells inside the biofilm by flow cytometry which revealed a significant decrease in the total number of cells when the feed water temperature was increased from 55 °C to 65 °C. Moreover, this process was accompanied by the permeate flux decline and increase of coolant water conductivity regardless of the spacer type. The results of our study have shown high rejection of dissolved organic carbon (DOC > 97%) and absence of bacterial contamination of permeate water which is important due to use of microporous polymeric membrane with 0.5 m pore size. The obtained results indicated the importance of operational conditions in controlling the biofouling in the MD system.