Long-term organic fertilizer (OF) application on agricultural soils is known to induce soil Cu and Zn contamination, along with pH and organic matter changes, which in turn alter the soil Cu and Zn availability. Our study was aimed at assessing Cu and Zn availability in long-term OF-amended soils by distinguishing the importance of increased contamination levels versus pH and organic matter changes in soil. Seventy-four soil samples were collected over time from fields corresponding to three soil types upon which no, mineral, or organic fertilization had been applied over a decade, and thus exhibited a gradient of Cu and Zn contamination, pH, and organic matter concentration. Soil Cu and Zn contamination (i.e. total and DTPA-extractable Cu and Zn concentration), soil solution chemistry (i.e. pH and dissolved organic matter concentration and aromaticity) and Cu and Zn availability (i.e. total concentration and free ionic activity in solution and DGT-available concentration in soil) levels were measured. The Windermere humic aqueous model (WHAM) was used to estimate Zn2+ activity and dissolved organic matter (DOM) binding properties in soil solution. Regardless of the soil type, organic fertilization increased Cu and Zn contamination in soil, in addition to the pH and the DOM concentration, aromaticity and binding properties in soil solution. The pH increase prompted a decrease in the total Zn concentration and Zn2+ activity in soil solution. The concomitant pH increase and DOM concentration, aromaticity and binding properties boosted the total Cu concentration but decreased the Cu2+ activity in soil solution. DGT-available Cu and Zn varied very little between the three fertilization modalities. Our results suggest that pH and DOM changes were able to regulate Cu and Zn availability in long-term OF amended soils by exerting a protective effect that offset the concomitant increase in soil Cu and Zn contamination.