Background and aims <p>Soil extracellular polymeric substances (EPS) are biopolymers secreted by microorganisms, primarily composed of polysaccharides (EPS-Poly), proteins (EPS-Prot), and DNA. These substances significantly influence soil physicochemical properties and are crucial for sustaining soil microbial activity. To analyze soil EPS, the cation exchange resin (CER) method, originally developed for wastewater EPS extraction, has been adapted to extract EPS from the soil matrix. However, the optimal experimental conditions for soil applications lack rigorous validation.</p> Methods <p>This study investigated the influence of varying CER additions and extraction durations on EPS yield and contamination across three distinct soil types. EPS content was quantified using colorimetric assays, while compositional variations were analyzed using Fourier-transform infrared spectroscopy and excitation-emission matrix analysis. The intracellular and extracellular contaminations were assessed by quantifying soil adenosine triphosphate (ATP) content post-extraction and humic substances in the extract, respectively. The optimal extraction conditions were identified via the technique for order preference by similarity to ideal solution analysis.</p> Results <p>Our findings reveal that increasing resin addition and extraction duration enhanced EPS-Poly, EPS-Prot, and DNA yields. However, soil ATP and humic substance analyses indicate concomitant increases in intracellular and extracellular contaminations. Furthermore, the technique for order preference by similarity to ideal solution (TOPSIS) analysis suggests that a 1&#xa0;mg SOC:178&#xa0;mg CER addition and a 2-hour extraction period achieve a balanced extraction efficiency while minimizing contamination.</p> Conclusions <p>Consequently, these previously established conditions derived from wastewater studies are effective for EPS extraction from these soils.</p>