The files in this data release (Kellogg and others, 2022) are those referenced in the journal article by Evans and others (2022) entitled “Biofilms as Potential Reservoirs of Stony Coral Tissue Loss Disease.” They contain an amplicon sequence variant (ASV) table and the raw 16S ribosomal ribonucleic acid (rRNA) gene amplicon deoxyribonucleic acid (DNA) sequence files from 15 microbial communities (sample names: CnD16B, CnD17B, CnD18B, CnD19B, CnD21B, CnD22B, CnD23B, CnD24B, CnD25B, PsD5B, CnH101B, CnH100B, Pstr4B, Pstr10B, and seawaterB) derived from biofilms on stainless steel plates (316 grade) formed in mesocosms containing diseased corals (stony coral tissue loss disease, n = 10), healthy corals (n = 4), or no coral (n = 1). Also included are the sequence files from a mock community, and two reagent blanks. Corals (Colpophyllia natans and Pseudodiploria strigosa) were collected between January 2019 and March 2021, from various locations throughout the Florida Keys. Plates were added to mesocosm buckets containing ultraviolet (UV)-sterilized, 0.2 micrometer (μm)-filtered seawater and either diseased (n = 10), healthy (n = 4), or no coral (n = 1) at the Smithsonian Marine Station in Fort Pierce, Florida (FL) on March 25, 2021. Plates were removed on March 28, 2021, and placed in individual sterile whirlpaks with RNAlater for future processing. Extraction of DNA from the samples and blanks occurred in April 2021 at the Coral Microbial Ecology Laboratory in St. Petersburg, FL using a QIAGEN DNeasy PowerBiofilm Kit. Library preparation and DNA sequencing were conducted on May 13, 2021, by the Michigan State University RTSF Genomics Core (East Lansing, Michigan) using primers 515F/806R to target the V4 variable region of the 16S rRNA gene. Sequencing was performed on an Illumina MiSeq sequencing system with v2 chemistry to obtain paired-end 250-bp (base pair) reads. The raw data files associated with this data release have also been submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under BioProject number PRJNA828575. For more information, please see the README file, README_Biofilm.txt.