Concerning the contribution of fungi to soil carbon sequestration, various methods have been used to extract ergosterol from soil samples. This study aims to explore the extraction ability and applicability of commonly used methods to extract ergosterol from two contrasting soils. An agricultural soil (chernozem) and a forest soil (podzol) were extracted with different types of cell lysis such as alkaline, glass bead, and ultrasonication methods in association with simple shaking. The ergosterol concentration was measured by high pressure liquid chromatography. Regardless of the method applied, ergosterol yield was higher in podzol than in chernozem. Alkaline extraction resulted in the highest ergosterol concentrations for both soils and miniaturized glass bead extraction produced comparable results in the case of chernozem. In terms of applicability, the non-alkaline methods were simpler to conduct and less demanding of labour, chemical use and glassware and more flexible in terms of the equipment used for mechanical disruption. Despite the limit of the two soil types in the present study, only the simple shaking method was revealed to be dependent on soil type. Based on our results, we recommend the miniaturized glass bead method for agricultural soils, low in organic matter for high throughput. However, not all of the methods described allow for the proper separation of co-extracted organic substances from organic-rich soil.