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Abstract Microbiome analysis through 16S rRNA gene sequencing is a crucial tool for understanding the microbial ecology of any habitat or ecosystem. However, workflows require large equipment, stable internet, and extensive computing power such that most of the work is performed far away from sample collection in both space and time. Performing amplicon sequencing and analysis at sample collection would have positive implications in many instances including remote fieldwork and point-of-care medical diagnoses. Here we present SituSeq, an offline and portable workflow for the sequencing and analysis of 16S rRNA gene amplicons using Nanopore sequencing and a standard laptop computer. SituSeq was validated by comparing Nanopore 16S rRNA gene amplicons, Illumina 16S rRNA gene amplicons, and Illumina metagenomes, sequenced using the same environmental DNA. Comparisons revealed consistent community composition, ecological trends, and sequence identity across platforms. Correlation between the abundance of taxa in each taxonomic level in Illumina and Nanopore data sets was high (Pearson’s r > 0.9), and over 70% of Illumina 16S rRNA gene sequences matched a Nanopore sequence with greater than 97% sequence identity. On board a research vessel on the open ocean, SituSeq was used to analyze amplicon sequences from deep sea sediments less than 2 h after sequencing, and 8 h after sample collection. The rapidly available results informed decisions about subsequent sampling in near real-time while the offshore expedition was still underway. SituSeq is a portable and user-friendly workflow that helps to bring the power of microbial genomics and diagnostics to many more researchers and situations.