The present study examines the effects of different fertilization treatments (chemical fertilization, farmyard manure, plant compost, and mycorrhiza-inoculated compost) on the soil fungi under a crop rotation of wheat (Triticum aestivum L.) and corn (Zea mays L.) in a long-term field experiment established in Mediterranean Turkey in 1996. Soil samples were collected in May, August, and October 2009. Soil pH, organic carbon, plant-available nitrogen and phosphorus, mycorrhizal colonization, and a series of biochemical markers (phospholipid and neutral lipid fatty acid [PLFA and NLFA] profiles, soil ergosterol content, and glomalin related soil protein [GRSP] as indicators of abundance of bacteria, saprotrophic, and arbuscular mycorrhizal [AM] fungi) were assessed. No significant difference was observed in soil organic C and plant available N in relation to long-term fertilization treatments, but plant available P in soil changed significantly in relation to the fertilization treatment used and the sampling season (between 11.5–33.8 mg · kg−1 in spring, 10.4–28.6 mg · kg−1 in summer, and 10.5–33.2 mg · kg−1 in autumn). Mycorrhizal colonization patterns were similar for both plants. However, mycorrhiza-inoculated compost treatment exhibited higher root colonization (77.3%) over control (16.3%), chemical fertilization (10.0%), farmyard manure (19.3%), and plant compost (20.0%). No statistically significant change was observed in ergosterol content. The effect of long-term organic treatments on soil PLFA structure was statistically prominent; whereas seasonality only affected bacterial PLFAs. Organic fertilization increased GRSP (mean annual ranging from 0.91 to 2.46 mg · g−1 total GRSP) but long-term annual mycorrhizal inoculation had no significant effect on the soil GRSP pool.