Conventional agriculture can lead to reduced soil organic matter and depletion in soil fertility. For that reason, Food and Agriculture Organization of the United Nations (FAO) recommends organic matter incorporation to soils to increase their agronomic quality. This work studies the effect of the transition to organic farming on chemical and biochemical properties of a loam soil (Xerofluvent), through a succession of five crops cycles over a 3-yr period. Two mature composts (vegetal and animal compost) were used for the organic fertilization. Soil chemical and biological status was evaluated by measuring total organic carbon (TOC), humic acids, bicarbonate-extractable P (Olsen-P), ammonium acetate extractable-potassium (AAE-K), Kjeldahl-N, microbial biomass carbon (MBC), microbial biomass nitrogen (MBN), enzymatic activities (dehydrogenase, protease, glucosidase, alkaline phosphatase), soil respiration, MBC/TOC, and MBC/MBN ratios. At the end of the study, the organically fertilized soils showed an increase in quantity (TOC) and quality (humic acids) of organic matter compared to inorganically fertilized soils. Nutrient content (Kjeldahl-N, Olsen-P, and AAE-K) also increased in organically fertilized soils. In general animal compost improved chemical and biological properties more than vegetal compost. Soil respiration was highly influenced by seasonal variability, and the highest values were found in summer. The MBC/TOC values indicated that microorganisms converted C to MBC more efficiently in conventionally fertilized soil. Protease and phosphatase activities differed between treatments after the third crop cycle, and the highest values were observed in organically fertilized soil. The TOC and nutrient content were correlated (P < 0.001) with microbial biomass and enzymatic activities. Extracellular enzyme activities (protease, glucosidase, alkaline phosphatase) were found to be significantly and positively correlated with MBC and MBN.