Soil extracts are routinely used to quantify dissolved organic nutrient concentrations in soil. Here we studied the loss and transformation of low molecular weight (LMW) components of DOC (14C-glucose, 1 and 100 μM) and DON (14C-amino acid mixture, 1 and 100 μM) during extraction of soil (0–6 h) with either distilled water or 0.5 M K2SO4. The extractions were performed at 20 °C, at 4 °C, or in the presence of an inhibitor of microbial activity (HgCl2 and Na-azide). We showed that both glucose and amino acids became progressively lost from solution with increasing shaking time. The greatest loss was observed in H2O extracts at 1 μM for both substances (>90% loss after 15 min). Lower temperature (4 °C) and presence of K2SO4 both resulted in reduced loss rates. The presence of microbial inhibitors effectively eliminated the loss of glucose and amino acids. We conclude that microbial transformation of LMW-DOC and DON during H2O or K2SO4 extraction of soil may affect the estimation of their concentrations in soil. This finding has significant implications for methods that rely on chemical extractions to estimate LMW-C components of DOC and DON. Research highlights ► Up to 90% of the added 14C-tracers (1 and 100 μM glucose and amino acids) were lost after as short as 15 min extraction. ► Lower temperature and presence of K2SO4 both resulted in reduced loss rates. ► Presence of microbial inhibitors effectively terminated the loss of glucose and amino acids. ► We conclude that microbial transformation of LMW-DOC and DON during soil extractions may affect the estimation of their composition in soil