The relationship among sugar concentrations, microbial community and physical variables (precipitation and soil temperature) was investigated in a ryegrass soil from January 2004 to January 2005. Mono- and disaccharide sugars were extracted using a mixture of dichloromethane and methanol and analyzed as their TMS derivatives by GC-MS. Changes in microbial community were assessed using phospholipid and neutral lipid fatty acids (PLFA and NLFA, respectively) analysis. The results of a one-year study showed that the seasonal variability of sugar contents found in the soil samples is mainly related to biomass or nutritional status of the fungal community. The increase in sucrose and fructose exudation by plant roots in the beginning of the growing season (early spring) may be responsible for the highest fungal biomass amount (PLFAs) observed in this study. Fungal storage lipid abundances (NLFAs) peaked in summer, during the same period that the highest concentrations of mannitol and trehalose were detected. This is consistent with these two sugars being stress-induced fungal metabolites, produced due to the low soil moisture observed during this season. In contrast, bacterial community growth seems to be more dependent on plant substrate than on physical variables, since the strongest decrease in bacterial biomass amounts (PLFAs) was found after cutting of the ryegrass field in early July.