Technological developments are a great advantage for biodiversity monitoring, if they are cross-validated with established protocols before their routine application. In the context of fish communities’ characterization, portable sequencers such as Oxford Nanopore Technology (ONT)’s MinION devices have the power to revolutionise field surveys through eDNA metabarcoding. However, Nanopore’s raw reads high error rates can disincentivise its use when compared to Illumina sequencers. Here, three sequencing protocols (two for Nanopore and one for Illumina) targeting short and long regions of two mitochondrial genes (12S and COI) were compared using fish mock communities and environmental DNA samples from mesocosm and field environments. Raw read quality, species richness detected, and taxonomic resolution of the platforms and genes were assessed. The latest Nanopore chemistry was comparable to Illumina for number of species retrieved, however discrepancies in species detected were observed between platforms and genes. Long amplicons provided higher taxonomic resolution compared to the short fragments. The 12S markers outperformed COI by detecting a higher number of species, while a multi-marker approach increased overall species richness in most cases. Sequencing technologies and primer selection can have significant impacts on fish eDNA metabarcoding results. Given its high specificity and taxonomic resolution, the long 12S region is presented as a promising marker for long-read metabarcoding. The latest Nanopore sequencing technology successfully profiled fish communities from mock and eDNA samples and due to its portability, quick turn-around time and reduced costs provides an innovative tool to conduct field-base studies.