ITSoneDB is a comprehensive collection of the fungal ribosomal RNA Internal Transcribed Spacer 1 (ITS1) sequences aimed at supporting metagenomic surveys of fungal environmental communities. The sequences were extracted from Genbank (GB) and arranged on the NCBI taxonomy tree. ITS1 start and end boundaries were defined by GB annotations and/or designed by mapping Hidden Markov Model (HMM) profiles of flanking 18S and 5.8S ribosomal RNA coding genes on each sequence.