16S amplicon sequencing is widely used for microbiome analysis but most bioinformatics tools cannot produce high quality genus-level or species-level taxonomic calls. This study evaluated the sensitivity and specificity of several bioinformatics pipelines and genomic reference libraries for taxonomic classification of 16S amplicon reads using 136 mock community samples. PathoScope 2 and Kraken 2, tools designed for whole-genome metagenomics, outperformed DADA2, QIIME 2, and Mothur, which are theoretically specialized for 16S analyses. Results support PathoScope and Kraken 2 as fully capable, competitive options for genus- and species-level 16S amplicon sequencing data analysis.