Although the resistance of sexually transmitted bacterium Chlamydia trachomatis was not of great concern due to its excellent sensitivity to standard antibiotic therapy, treatment failures associated with laboratory-confirmed resistance have been described in the literature. Croatia is a country with a high rate of antimicrobial drug prescriptions which can give rise to resistant or less sensitive chlamydial strains. Due to the laborious methodology, chlamydial isolates so far have not been subjected to antimicrobial susceptibility testing in our country. The aim of this study was to evaluate in vitro efficacy of azithromycin, doxycycline and levofloxacin for 40 urogenital chlamydial strains isolated from Croatian patients over a period of three years. 23 isolates from cervical swabs, 16 isolates from male urethral swabs, 1 isolate from expressed prostatic secretions and 2 reference strains were subjected to antimicrobial susceptibility testing on McCoy cells. Minimal inhibitory concentration (MIC) as the lowest concentration of antibiotic without microscopically visible inclusions and minimal chlamydicidal concentration (MCC) as the concentration without visible inclusions after one passage in antibiotic-free cell culture have been determined for each strain. All strains were susceptible to azithromycin, doxycycline and levofloxacin in vitro (MIC < 4 μg/ml), hence “homotypic” or “heterotypic” resistance pattern has not been found in any of the tested isolates. MCC values were equal to MIC values, or were one to five dilutions higher. For all strains together MIC ranged from 0.064 to 0.125 μg/ml for azithromycin, from 0.016 to 0.125 μg/ml for doxycycline, and from 0.125 to 1.0 μg/ml for levofloxacin; MCC ranged from 0.064 to 2.0 μg/ml for azithromycin, from 0.032 to 1.0 μg/ml for doxycycline, and from 0.125 to 2.0 μg/ml for levofloxacin. Concentrations of tested reference strains (serovar E Bour and serovar F ICCal) were in concordance with medians of urogenital chlamydial strains from Croatian patients, or were one dilution higher. Comparison of MIC and MCC values of antimicrobial drugs revealed statistically significant difference in their in vitro efficacy. The strongest antimicrobial effect was shown by doxycycline, followed by azithromycin, whilst levofloxacin was the least effective. Our in vitro results are in accordance with the results of clinical research in our country and abroad, although we lack studies that directly compare the efficacy of levofloxacin with the effectiveness of azithromycin and doxycycline in the treatment of chlamydial infections in vivo. Statistically significant correlation was found for MCC in the case of two pairs of antimicrobials: azithromycin and levofloxacin, and doxycycline and levofloxacin. In both of those cases strains that require higher concentrations of azithromycin or doxycycline for chlamydicidal effect also necessitate a higher concentration of levofloxacin. Comparison of medians for different clinical samples did not show any statistically significant difference. Although resistant strains have not been found, certain isolates exhibited a significantly higher concentration values. The value of MCC for azithromycin was 1 μg/ml or higher in 29.4% of isolates from male urethral swabs and expressed prostatic secretions, i.e. in 15% of all the tested isolates, which is relevant when compared to the available literature. Since we still lack information on whether C. trachomatis is evolving in vivo in response to selection pressure of antimicrobial treatment, it is essential to continue with this kind of resistance surveillance – particularly in countries with high antibiotic consumption.