In June 2019, the Thünen Institute conducted the WH429 research cruise with the aim of assessing marine biodiversity through environmental DNA (eDNA) analysis. During the expedition, eDNA samples were collected from various marine locations and subsequently processed for metabarcoding using the MiFish primers, which target a specific region of the mitochondrial 12S rRNA gene in fish. The resulting eDNA sequences were generated on the Illumina MiSeq platform, producing high-quality fastq files. This dataset provides critical metadata, including sequencing reads, quality scores, which are essential for studying the spatial and temporal patterns of fish diversity. The comprehensive eDNA dataset serves as a valuable resource for ecological and conservation research, enabling the identification of fish species and the monitoring of marine ecosystems with high resolution and accuracy.