Biofouling is a hurdle of seawater desalination that increases water costs and energy consumption. In membrane distillation (MD), biofouling development is complicated due to the temperature effect that adversely affects microbial growth. Given the high relevance of MD to regions with abundant warm seawater, it is essential to explore the biofouling propensity of microbial communities with higher tolerance to elevated temperature conditions. This study presents a comprehensive analysis of the spatial and temporal biofilm distribution and associated membrane fouling during direct contact MD (DCMD) of the Red Sea water. We found that structure and composition of the biofilm layer played a significant role in the extent of permeate flux decline, and biofilms that built up at 45°C had lower bacterial concentration but higher extracellular polymeric substances (EPS) content as compared to biofilms that formed at 55 °C and 65°C. Pore wetting and bacterial passage to the permeate side were initially observed but slowed down as operating time increased. Intact cells in biofilms dominated over the damaged cells at any tested condition emphasizing the high adaptivity of the Red Sea microbial communities to elevated feed temperatures. A comparison of microbial abundance revealed a difference in bacterial distribution between the feed and biofilm samples. A shift in the biofilm microbial community and colonization of the membrane surface with thermophilic bacteria with the feed temperature increase was observed. The results of this study improve our understanding of biofouling propensity in MD that utilizes temperature-resilient feed waters.