Koala numbers in Eastern Australia have declined dramatically over the last 20 years. Over this time an increase in infectious diseases in the species, including chlamydia, has been observed. Chlamydia pecorum infection is estimated to effect approximately 70% of koalas in SEQ. In females, infection of koalas has shown to induce reproductive tract pathology resulting in infertility. To date, minimal studies have investigated the effects of C. pecorum infection on male fertility. This study was undertaken to investigate whether naturally occurring chlamydia-infection identified in koala semen is viable to inoculate a cell line and act as a source of bacterial transmission. Semen collected from 120 wild koalas were assessed for motility and rate, % live/dead, concentration, morphology, % DNA fragmentation and chlamydial speciation and quantitation determined through qPCR of seminal fluid. Chlamydia-positive koala semen, with PCR quantitated infectious load, was inoculated into a clean McCoy cell line and incubated. Fluorescently-conjugated chlamydia MOMP antibody was used for the detection of chlamydial inclusion bodies within cell cytoplasm. Results show inoculation of a cell line using naturally-infected koala semen. Observation of small chlamydial inclusion bodies and remnants of sperm flagella within the culture medium indicates presence of infection. With the absence of experimental animals available, we have developed a secondary method that can assist in determining the infectious load of chlamydia within a koala semen sample. Results confirm that infectious chlamydial elementary bodies can be shed into koala semen and serve as a source of bacterial transmission to the female.