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Oral swabs are emerging as a non-invasive sample type for diagnosing infectious diseases including Ebola, tuberculosis (TB), and COVID-19. To assure proper sample collection, sample adequacy controls (SACs) are needed that detect substances indicative of samples collected within the oral cavity. This study evaluated two candidate SACs for this purpose. One detected representative oral microbiota (Streptococcus species DNA) and the other, human cells (human mitochondrial DNA, mtDNA). Quantitative PCR (qPCR) assays for the two target cell types were applied to buccal swabs (representing samples collected within the oral cavity) and hand swabs (representing improperly collected samples) obtained from 51 healthy U.S. volunteers. Quantification cycle (Cq) cutoffs that maximized Youden's index were established for each assay. The streptococcal target at a Cq cutoff of ≤34.9 had 99.0% sensitivity and specificity for oral swab samples, whereas human mtDNA perfectly distinguished between hand and mouth swabs with a Cq cutoff of 31.3. The human mtDNA test was then applied to buccal, tongue, and gum swabs that had previously been collected from TB patients and controls in South Africa, along with "air swabs" collected as negative controls (total N = 292 swabs from 71 subjects). Of these swabs, 287/292 (98%) exhibited the expected Cq values. In a paired analysis the three oral sites yielded indistinguishable amounts of human mtDNA, however PurFlockTM swabs collected slightly more human mtDNA than did OmniSwabsTM (p = 0.012). The results indicate that quantification of human mtDNA cannot distinguish swabs collected from different sites within the mouth. However, it can reliably distinguish oral swabs from swabs that were not used orally, which makes it a useful SAC for oral swab-based diagnosis. [ABSTRACT FROM AUTHOR]