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The study of fungi in marine sediments has received a growing attention in the recent years. Recent advances in metagenomics have allowed the discovery of a wide diversity of fungi in deep-sea environments, although standardized methods are needed to process the samples and to analyze the data. This protocol is a guidance to obtain good quality samples and subsequent ITS1 (Internal Transcribed Spacer 1) amplicons to be prepared for sequencing by Illumina platform. The collection of marine sediments is described, as well as the processing of the samples in laboratory; also, pertinent controls that should be included during all the processing steps to identify potential sources of contamination that could affect the sequencing and bias the interpretation of results are suggested.