The accurate detection of a great number of microorganisms within a stool sample is critical to initiate the appropriate treatment in a clinical setting. Here we have shown that 16S rRNA gene sequencing can accurately detect and quantify clinically relevant levels of 28 target bacteria and archaea. We demonstrate that many prokaryotic targets identified from the literature as associated with human health can be consolidated in an assay, and further that relating the relative levels of bacteria and archaea to a healthy reference range enables the reporting of positive results only when clinically relevant.