Previously, we reported that a compound from a group of thiohydrazides of oxamic acids, CL-55, possessed antichlamydial activity in vitro that was accompanied by a decreased translocation of the type three secretion effector, IncA, into the host cell. In this study, the antichlamydial activity of CL-55 was tested in vivo in DBA/2 mice infected with C. trachomatis serovar D. We found that intravaginal inoculation of DBA/2 mice with the clinically relevant strain, C. trachomatis serovar D, results in a course of infection and pathology similar to that observed in humans. The early stage of infection in this model was characterized by a shedding of Chlamydia in vaginal secretions followed by an ascending infection and inflammation in the upper genital tract. We found that CL-55 possessed antibacterial activity in vivo and was able to control C. trachomatis vaginal shedding, ascending infection, and inflammation in the upper genital organs in DBA/2 mice. Our data provide a proof of concept for the protective effect of the thiadiazinon, CL-55, against chlamydial infection in vivo and support the feasibility of further studies of its potential therapeutic applications.