A laboratory experiment was performed to examine the medium-term influence of three tetracycline antibiotics (chlortetracycline, CTC; tetracycline, TC and oxytetracycline, OTC) at different concentrations in four agricultural soils with similar pH and different soil organic content. After a 42-days incubation period, three different soil enzymes (β-glucosidase, urease, and phosphomonoesterase) were estimated, as well as the phospholipid fatty acids (PLFAs). A residual effect was observed on all microbial parameters measured in the four soils affecting to the soil enzymes activity and soil microbial communities structure (PLFA pattern). A different microbial sensitivity to antibiotics was detected depending on both, soil type and the microbial property considered. Specifically, in general, no antibiotic effect or even a slight positive effect was observed for phosphomonoesterase and β-glucosidase enzyme activities, respectively, while a negative effect was detected for urease activity values, particularly at higher doses of the antibiotics in a soil with a low organic matter content. The principal component analysis performed with the PLFAs data obtained for all soil samples showed different microbial communities depending mainly on soil type, followed by the antibiotic added to the soil (CTC, TC or OTC) and, in a lesser extent, by its concentration. In general, the PLFA patterns showed similar microbial communities structure due to OTC and TC addition in comparison to the microbial communities structure of soil treated with CTC. These results could be environmentally relevant, especially as regards potential effects of antibiotics on the soil microbiome and hence on health risk assessment of these antibiotics in soils.