Problem Chlamydial infections represent a major threat to the survival of the koala. Infections caused by Chlamydia pecorum cause blindness, infertility, pneumonia and urinary tract infections and represent a threat to the survival of the species. Little is known about the immune response in koalas, or the safety of commonly used adjuvants for induction of protective systemic and mucosal immunity. Method of study In the present study, we immunized 18 healthy female koalas subcutaneously with a combination of three chlamydial antigens [major outer membrane protein (MOMP), NrdB and TC0512 (Omp85)] mixed with one of three different adjuvants [Alhydrogel, Immunostimulating Complex (ISC) and TiterMax Gold]. Results All adjuvants induced strong neutralizing IgG responses in plasma against the three antigens with prolonged responses lasting more than 270 days seen in Alhydrogel and ISC immunized animals. Cloacal IgG responses lasting >270 days were also induced in ISC-immunized animals. Chlamydia-specific peripheral blood mononuclear cell proliferative responses were elicited by both Alhydrogel and ISC, and these lasted >270 days in the ISC group. Conclusion The data show that a multi-subunit chlamydial vaccine, given subcutaneously, can elicit Chlamydia-specific cell-mediated and antibody responses in the koala demonstrating that the development of a protective vaccine is feasible.