The deepest point of the oceans is over 10 km depth and is characterized with extremely high hydrostatic pressure. Water sampling from the hadal zone for microbiological studies is therefore difficult. In this study, we demonstrate the application of an in situ microbial filtration and fixation (ISMIFF) apparatus in sampling work from the Challenger Deep. A total of 2300 L hadal waters were filtered by the ISMIFF equipped on a hadal lander during seven deployments. The microbial communities in the hadal samples ( > 6000 m) collected by the ISMIFF were composed of more heterotrophic Marinimicrobia and less photoautotrophic Prochlorococcus and SAR11 bacteria, compared with those by Niskin bottles. Such a difference in the community structure was not observed in the samples collected by the two methods from 5000-m depth, indicating an effect of greater depths on sample quality. Our results indicate that the ISMIFF or similar filtration apparatuses are indispensable for hadal water sampling since using Niskin bottles is probably difficult to obtain pristine samples from the hadal zone. Application of the ISMIFF will pave the way to meta-omics studies on the hadal microbes.