Relatively little information is available for the composition of microbial communities present in hadal environments, the deepest marine locations. Here we present a description of the phylogenetic diversity of particle-associated (> 3 µm) and free-living (3–0.22 µm) microorganisms present in a pelagic trench environment. Small subunit ribosomal RNA gene sequences were recovered from members of the Bacteria, Archaea and Eukarya obtained from a depth of 6000 m in the Puerto Rico Trench (PRT). Species richness estimates for the bacterial particle-associated fraction were greater compared with the free-living fraction and demonstrated statistically significant compositional differences, while the archaeal fractions were not found to be significantly different. The particle-associated fraction contained more Rhodobacterales and unclassified Myxococcales along with Bacteroidetes, Planctomycetes and chloroplast sequences, whereas the free-living fraction contained more Caulobacterales, Xanthomonadales and Burkholderiales, along with Marine Group A and Gemmatimonadetes. The Eukarya contained a high abundance of Basidiomycota Fungi 18S rRNA genes, as well as representatives from the super-groups Rhizaria, Excavata and Chromalveolata. A diverse clade of diplonemid flagellates was also identified from the eukaryotic phylotypes recovered, which was distinct from previously identified deep-sea pelagic diplonemid groups. The significance of these results to considerations of deep-sea microbial life and particle colonization is discussed in comparison to the few other deep-ocean phylogenetic surveys available.