The aim of the study was to evaluate the changes in glomalin-related soil proteins (GRSP) content, microbial diversity and soil physical quality depending on the type of soil measures of soil improvement and changes in soil health. The study was based on a 100-year stationary field microplot experiment where the soil profiles were collected with preserving the natural soil horizons. The microplot experiment was carried out on eight different soil types: Brunic Arenosol (Dystric I), Rendzic Leptosol, Fluvic Cambisol, Haplic Cambisol (Eutric), Gleyic Phaeozem, Brunic Arenosol (Dystric II), Haplic Cambisol (Eutric II) and Haplic Cambisol (Dystric). These soils are the most common types of agricultural soils in Poland. Relatively significant correlations with the soil quality, physical parameters and the glomalin-related soil proteins have been found. The study determined the total GRSP (T-GRSP) and easily extractable GRSP (EE-GRSP) levels in soils as well as the soil physical quality index and soil’s microbial biodiversity. The GRSP depended on the type of soil and correlated with S-Index and also was responsible for the unique chemical and physical properties of soils. Soils characterized by the highest T-GRSP content belonged to the group of very good and good soil physical quality characterized also by high biological activity, for which there were strong correlations with such parameters as dehydrogenase activity (DHA), microbial biomass content (MBC), microbial nitrogen content (MBN) and total bacteria number (B). The highest T-GRSP content and higher microbial diversity were found in Gleyic Phaeozem, Rendzic Leptosol and Fluvic Cambisol. The T-GRSP and EE-GRSP content were additionally correlated with the number of AMF spores. Very poor and poor soil physical quality according to S-Index characterized Brunic Arenosol (Dystric I) and Haplic Cambisol (Dystric). This research indicates that a specific edaphone of soil microorganisms and GRSP content may be of great importance when assessing a soil’s quality and improvements in soil health. The abundance of glomalin-producing fungi significantly affects the quality of the soil. This effect is particularly important for agricultural soils are threatened by ongoing land degradation.