Two field experiments, in the south of Italy, were established in 2009 to study and characterize soil organic matter (SOM) during transition to organic farming. Experiments included a cereal/leguminous rotation fertilized with permitted amendments with three field replicates. A sequential fractionation procedure was used to separate different SOM fractions: light fraction (LF), two size classes of particulate organic matter (POM), mobile humic acid (MHA), and Ca-bound humic acid (CaHA). Isolated fractions were quantified and analyzed for their C and N content and carbohydrate and amino compound composition. The masses of the isolated fractions increased during 2-year course, with noticeable increases in LF and POM. Moreover, LF and POM were found more responsive than MHA to treatment and crop. The xylose/mannose ratio explained that MHA-carbohydrates were mainly of microbial origin, while LF- and POM-carbohydrates were of plant origin. Amino compounds constituted up to 30% of total soil N and were found to be more responsive to seasonal variation than to agronomic practices.