The objective of this study was to determine the effect of antibiotics on Chlamydia trachomatis viability by using a quantitative real-time PCR assay that measured DNA replication and mRNA transcription of the structural omp1 and omp2 genes, 16S rRNA, and the groEl1 gene with and without antibiotics. Ofloxacin, moxifloxacin, azithromycin, and doxycycline were tested against the serovar D and L2 reference strains and a derivative mutant resistant to fluoroquinolones L2-OFXR obtained by in vitro selection. By using DNA quantification, the antibiotic MIC was calculated when the number of DNA copies was equal to that of the chlamydial inoculum at T0. This method allowed an easy determination of the MIC by DNA quantification of the four selected genes and gave similar results to those obtained by immunofluorescence staining without biased interpretation. By using cDNA quantification, the lowest antibiotic concentration for which no RNA was transcribed corresponded to the minimum bactericidal concentration. C. trachomatis still transcribed 16S rRNA or groEl1 genes even at concentrations well above the MIC, showing a bacteriostatic effect for all antibiotics tested. This method allows the study of antibiotic activity on growth and viability of C. trachomatis by DNA and RNA quantification at the same time without additional cell culture passaging.