Chlamydia trachomatis and Waddlia chondrophila are strict intracellular bacteria belonging to the Chlamydiales order. C. trachomatis is the most frequent bacterial cause of genital and ocular infections whereas W. chondrophila is an opportunistic pathogen associated with adverse pregnancy outcomes and respiratory infections. Being strictly intracellular, these bacteria are engaged in a complex interplay with their hosts to modulate their environment and create optimal conditions for completing their life cycle. For this purpose, they possess several secretion pathways and, in particular, a Type III Secretion System (T3SS) devoted to the delivery of effector proteins in the host cell cytosol. Identifying these effectors is a crucial step in understanding the molecular basis of bacterial pathogenesis. Following incubation of infected cells with perfringolysin O, a pore-forming toxin that binds cholesterol present in plasma membranes, we analysed by mass spectrometry the protein content of the host cell cytoplasm. We identified 13 putative effectors secreted by C. trachomatis and 19 secreted by W. chondrophila. Using Y. enterocolitica as a heterologous expression and secretion system, we confirmed that four of these identified proteins are secreted by the T3SS. Two W. chondrophila T3SS effectors (hypothetical proteins Wcw_0499 and Wcw_1706) were further characterised and demonstrated to be early/mid-cycle effectors. In addition, Wcw_1706 is associated with a tetratricopeptide domain-containing protein homologous to C. trachomatis class II chaperone. Furthermore, we identified a novel C. trachomatis effector, CT460 that localises in the eukaryotic nucleus when ectopically expressed in 293 T cells.