Although Chlamydia trachomatis resistance is not of great concern due to its excellent sensitivity to the currently recommended first-line antibiotics (azithromycin and doxycycline), clinical treatment failures have been reported and some of them were linked to laboratory proved resistance. The aim of this study was to determine in vitro susceptibility to azithromycin and doxycycline for 24 urogenital chlamydial strains isolated in Croatia-a country with the highest consumption of azithromycin in Europe and with very high antibiotic prescription rates. Fourteen isolates from cervical swabs, nine from male urethral swabs, and one isolate from expressed prostatic secretion were tested in McCoy cell culture system. All strains were susceptible to azithromycin and doxycycline with minimal inhibitory concentration for azithromycin and doxycycline ranging from 0.064 to 0.125 μg/mL and 0.016 to 0.064 μg/mL, and minimal chlamydicidal concentration ranging from 0.064 to 2.0 μg/mL and 0.032 to 1.0 μg/mL, respectively. Since we still lack information on whether C. trachomatis is evolving in vivo in response to antibiotic selection pressure, this kind of surveillance for resistance is essential in detecting shifts in antimicrobial susceptibilities.