A laboratory experiment was carried out to investigate the response of the microbial communities in acid agricultural soils located in the NW Iberian Peninsula to the presence of clarithromycin. Four soils, with different organic C content and similar pH, and seven different concentrations of clarithromycin (0.49, 1.95, 7.81, 31.25, 125, 500 and 2,000 mg kg−1 of soil) were used, and microbial estimates were made after 8 and 42 incubation days. The phospholipid fatty acids (PLFA) technique was used to estimate the total microbial biomass and biomass of specific microbial groups as well as the microbial community structure (PLFA pattern). The microbial biomass (total and specific groups) was different in the four studied soils, the lowest values being exhibited by soils with the lowest organic C. The antibiotic addition showed a positive effect on microbial biomass (total and specific groups), especially at the highest dose; the effect being similar or even more accentuated with time passed after the addition (42 days ≥8 days). Principal component analysis (PCA) of the PLFA data carried out with the whole data set showed that the main determining factors of the microbial structure followed the order: soil > time incubation ≥ antibiotic dose. When the PCA was performed individually for each incubation time, the results indicated that microbial communities of the four soils were different. Likewise, for each soil, different microbial communities were observed depending on antibiotic concentration. The microbial biomass and PLFA pattern data were coincidentally showing that the clarithromycin addition favored fungi and G− bacteria more that bacteria and G+ bacteria; the effect being dose-dependent. Our data (microbial biomass, PLFA pattern) also demonstrated that the effect of clarithromycin addition on microbial communities in these four acid agricultural soils persisted even after 42 incubation days.