This work examines the results of a soil incubation experiment in the laboratory, under controlled conditions of humidity and temperature. The purpose was to determine the medium-term influence of the presence of antibiotics on the total and specific microbial biomass, determined by means of the phospholipid fatty acids (PLFAs) analysis (total microbial biomass, and specific fungal, bacterial, actinobacterial, Gram-negative bacterial and Gram-positive bacterial biomass), as well as the relationship between some of these groups (fungal biomass/bacterial biomass, Gram-negativebacterial /Gram-positive bacterial). The experiment was performed with four different cultivated soils with a similar pH but different organic matter (OM) content, to which eight doses of three antibiotics of the tetracycline group (tetracycline, oxytetracycline and chlorotetracycline) were added. Microbial biomass measurements (total and specific groups) were performed after 42 days of incubation. As expected, the total and specific microbial biomass values were different in the four soils studied. Both the total and the specific microbial biomass showed a similar response to the presence of antibiotics, although in several cases the data were inconsistent and difficult to interpret. In general, in all soils the addition of chlorotetracycline and tetracycline slightly modified or increased, to a greater or lesser extent, the values of both total and specific microbial biomass, particularly at higher doses. However, in certain cases, biomass values decreased due to the addition of the highest dose of oxytetracycline.