We examined fungal communities in soil profiles of 0–10 cm depth along the altitudinal gradient of 250–530–990 m.a.s.l. at the Central Negev Desert, Israel, which benefit from similar annual precipitation (95 mm). In the soil samples collected in the summer of 2020, a mycobiota accounting for 169 species was revealed by both culture-dependent and culture-independent (DNA-based) methodologies. The impact of soil depth on the variations in fungal communities was stronger than the impact of altitude. Both methodologies displayed a similar tendency in the composition of fungal communities: the prevalence of melanin-containing species with many-celled large spores (mainly Alternaria spp.) in the uppermost layers and the depth-wise increase in the proportion of light-colored species producing a high amount of small one-celled spores. The culturable and the DNA-based fungal communities had only 13 species in common. The differences were attributed to the pros and cons of each method. Nevertheless, despite the drawbacks, the employment of both methodologies has an advantage in providing a more comprehensive picture of fungal diversity in soils.