<i>Waddlia</i>   <i>chondrophila</i> and <i>Simkania negevensis</i> are emerging <i>Chlamydia-</i> related bacteria. Similar to the pathogenic organisms <i>Chlamydia pneumoniae</i> and <i>Chlamydia trachomatis,</i> these emerging bacteria are implicated in human genital infections and respiratory diseases. We used a screening strategy based on a newly developed <i>S. negevensis</i> -specific quantitative real-time PCR (qPCR) and a pan- <i>Chlamydiales</i> qPCR. We could not detect <i>S. negevensis</i> in 458 respiratory, genitourinary, cardiac and hepatic samples tested. One urethral swab was positive for <i>W. chondrophila.</i> We observed a low prevalence of <i>Chlamydiales</i> in respiratory samples (1/200, 0.5%), which suggests that <i>C. pneumoniae</i> is an uncommon respiratory pathogen. Furthermore, we screened 414 human serum samples from Switzerland, England and Israel and observed a low prevalence (<1%) of exposure to <i>S. negevensis.</i> Conversely, humans were commonly exposed to <i>W. chondrophila,</i> with seroprevalences ranging from 8.6% to 32.5%. <i>S. negevensis</i> is not a clinically relevant pathogen, but further research investigating the role of <i>W. chondrophila</i> is needed.