The long‐term effect of inorganic and organic fertilization in a vegetable crop rotation on soil chemical and biochemical properties was investigated in a trial in southern Spain. Two crops were grown in succession, potato (Solanum tuberosum L.) and a mixed‐cropped strawberry–onion (Fragaria vesca L.–Allium cepa L.). Total organic carbon (TOC), Kjeldahl‐N, bicarbonate‐extractable P (Olsen‐P), ammonium acetate extractable‐potassium (AAE‐K), microbial biomass carbon (Cmic), and enzyme activity (dehydrogenase, β‐glucosidase, protease, and alkaline phosphatase) were determined in soils in the seventh and eighth year of management comparison. The metabolic quotient (qCO2) and the Cmic to TOC ratio were also calculated. Organically fertilized soils showed significant increases in TOC and Kjeldahl‐N, available‐P, AAE‐K, microbial biomass C, and enzymatic activities compared with those found under inorganically fertilized soils. The qCO2 values were greater in inorganic than in organic fertilized plots indicating a lower microbial community respired at a greater rate in inorganic fertilized soils. The Cmic to TOC ratio in organic plots was lower than in inorganic plots indicating that microorganism in inorganically fertilized soils could have a better efficiency in the conversion of C sources to microbial biomass. Long‐term organic fertilization positively affected soil organic matter content, thus improving soil chemical and biological fertility under arid environmental conditions in southwest Spain. However precautions must be taken as excessive accumulation of some nutrients, particularly N and P, can arise from the long‐term use of compost.