Microbiome sequencing is at the forefront of health management development, and as such, it is becoming of great interest to monitor the microbiome in the aquaculture industry as well. Oxford Nanopore Technologies (ONT) platforms are gaining popularity to study microbial communities, enabling faster sequencing, extended read length, and therefore, improved taxonomic resolution. Despite this, there is a lack of clear guidelines to perform a metabarcoding study, especially when dealing with samples from non-mammalian species, such as aquaculture-related samples. In this article, we provide general guidelines for sampling, nucleic acid extraction, and ONT-based library preparation for both environmental (water, sediment) and host-associated (gill or skin mucus, skin, gut content, or gut mucosa) microbiome analysis. Our procedures focus specifically on rainbow trout (Oncorhynchus mykiss) reared in experimental facilities. However, these protocols can also be transferred to alternative types of samples, such as environmental DNA (eDNA) monitoring from alternative water sources, or to different fish species. The additional challenge posed by the low biomass and limited bacterial diversity inherent in fish-associated microbiomes is addressed through the implementation of troubleshooting solutions. Furthermore, we describe a bioinformatic pipeline starting from raw reads and leading to taxonomic abundance tables using currently available tools and software. Finally, we provide a set of specific guidelines and considerations related to the strategic planning of a microbiome study within the context of aquaculture. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Environmental sample collection Basic Protocol 2: Host-associated sample collection Alternate Protocol: Host-associated sample collection: Alternative sample types Basic Protocol 3: Sample pre-treatment and nucleic acid extraction Basic Protocol 4: Quality control and preparation for 16S rRNA gene sequencing Support Protocol 1: Assessment of inhibition by quantitative PCR Support Protocol 2: Bioinformatic analysis from raw files to taxonomic abundance tables.