The MiFish pipeline works on the Linux/Unix environment. In the first step, the pipeline conducts quality-filtering of the MiFish sequence reads, including Phred score-based low-quality tail-trimming, length filtering, deletion of N base-containing reads, paired-end read assembly, and primer sequences removal. The fasta files of the reads that passed through these quality check are generated. As the next step, the pipeline performs sequence similarity-based species-assignment and the generation of a species by read-count table. This tab-spaced table provides species diversity information detected from the MiFish PCR meta-barcoding analysis of environmental DNAs.