This research examined the general soil fungi and AM fungal communities associated with a Lonely Tree species (Vachellia pachyceras) existing in the Sabah Al-Ahmad Natural Reserve located at the Kuwait desert. The goals of the study were to describe the general fungal and AM fungal communities present in the rhizospheric, non-rhizospheric soils and roots of V. pachyceras, respectively, as well as local and non-local V. pachyceras seedlings when grown under standard nursery growing environments. Soil and root samples were analyzed for an array of characteristics including soil physicochemical composition, and culture-independent method termed PCR-cloning, intermediate variable region of rDNA, the large subunit (LSU) and internal transcribed spacer (ITS) region sequence identifications. The results reveal that the fungal phylotypes were classified in four major fungal phyla namely Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. The largest assemblage of fungal analyses showed communities dominated by members of the phylum Ascomycota. The assays also revealed a wealth of incertae sedis fungi, mostly affiliated to uncultured fungi from diverse environmental conditions. Striking difference between rhizosphere and bulk soils communities, with more fungal diversities and Operational Taxonomic Units (OTUs) richness associated with both the field and nursery rhizosphere soils. In contrast, a less diverse fungal community was found in the bulk soil samples. The characterization of AM fungi from the root system demonstrated that the most abundant and diversified group belongs to the family Glomeraceae, with the common genus Rhizophagus (5 phylotypes) and another unclassified taxonomic group (5 phylotypes). Despite the harsh climate that prevails in the Kuwait desert, studied roots displayed the existence of considerable number of AM fungal biota. The present work thus provides a baseline of the fungal and mycorrhizal community associated with rhizosphere and non-rhizosphere soils and roots of only surviving V. pachyceras tree from the Kuwaiti desert and seedlings under nursery growing environments.