ABSTRACTChlamydia trachomatis
is a medically important human pathogen causing different diseases, including trachoma, the leading cause of preventable blindness in developing countries, and sexually transmitted infections that can lead to infertility and ectopic pregnancies. There is no vaccine against
C. trachomatis
at present. Broad-spectrum antibiotics are used as standard therapy to treat the infection but have unwanted side effects, such as inducing persistent or recurring infections and affecting the host microbiome, necessitating the development of novel anti-
Chlamydia
therapies. Here, we describe the establishment of a robust, fast, and simple plaque assay using liquid overlay medium (LOM) for the identification of anti-
Chlamydia
compounds. Using the LOM plaque assay, we identified nitrobenzoxadiazole (NBD)-labeled 1-
O
-methyl-ceramide-C
16
as a compound that efficiently inhibits
C. trachomatis
replication without affecting the viability of the host cell. Further detailed analyses indicate that 1-
O
-methyl-NBD-ceramide-C
16
acts outside the inclusion. Thereby, 1-
O
-methyl-NBD-ceramide-C
16
represents a lead compound for the development of novel anti-
Chlamydia
drugs and furthermore constitutes an agent to illuminate sphingolipid trafficking pathways in
Chlamydia
infections.