STUDY QUESTION: What is the impact of Waddlia chondrophila, an emerging Chlamydia-related bacterium associated with miscarriage, on human spermatozoa? SUMMARY ANSWER: W. chondrophila had a negative impact on human spermatozoa (decrease in viability and mitochondrial membrane potential) and was not entirely removed from infected samples by density gradient centrifugation. WHAT IS KNOWN ALREADY: Bacterial infection or colonization might have a deleterious effect on male fertility. Waddlia chondrophila was previously associated with miscarriage, but its impact on male reproductive function has never been studied. STUDY DESIGN SIZE, DURATION: An in vitro model of human spermatozoa infection was used to assess the effects of W. chondrophila infection. Controls included Chlamydia trachomatis serovar D and latex beads with similar size to bacteria. PARTICIPANTS/MATERIALS, SETTING, METHODS: Purified motile spermatozoa were infected with W. chondrophila (multiplicity of infection of 1). Immunohistochemistry combined with confocal microscopy was used to evaluate how bacteria interact with spermatozoa. The impact on physiology was assessed by monitoring cell viability, mitochondrial membrane potential and DNA fragmentation. MAIN RESULTS AND THE ROLE OF CHANCE: Using super-resolution confocal microscopy, bacteria were localized on spermatozoa surface, as well as inside the cytoplasm. Compared to controls, W. chondrophila caused a 20% increase in mortality over 72 h of incubation (P < 0.05). Moreover, higher bacterial loads significantly reduced mitochondrial membrane potential. Bacteria present on spermatozoa surface were able to further infect a cell-monolayer, indicating that sperm might vector bacteria during sexual intercourse. LIMITATIONS REASONS FOR CAUTION: The main limitation of the study is the use of an in vitro model of infection, which might be too simplistic compared to an actual infection. An animal model of infection should be developed to better evaluate the in vivo impact of W. chondrophila. WIDER IMPLICATIONS OF THE FINDINGS: Intracellular bacteria, including C. trachomatis, Mycoplasma spp. and Ureaplasma spp., are associated with male infertility. Waddlia chondrophila might represent yet another member of this group, highlighting the need for more rigorous microbiological analysis during investigations for male infertility. STUDY FUNDING/COMPETING INTEREST(S): This work has been funded by the Department of Obstetrics and Gynecology, Lausanne University Hospital, Switzerland, and by the Swiss National Science Foundation (Grant nos. 310030-156169/1, 320030-169853/1 and 320030-169853/2 attributed to D.B.). D.B. is also supported by the -Fondation Leenaards' through the -ourse pour la relve academique', by the -Fondation Divesa' and by the -Loterie Romande'. No conflicts of interest to declare.