Functional structure and diversity of soil free-living nematodes in a desert environment depend on plant gender and sampling site. The objective of this study was to compare the composition, abundance and tropic group of soil free-living nematodes in the upper 0–10 cm soil layer under the male and female Acanthosicyos horridus Welw. ex Hook. f. plants and in the inter-shrub open areas (control) in the Namib Desert, Namibia in April 2015. Soil moisture, organic matter (OM) and pH was also analyzed. Free-living nematodes were extracted from 100 g soil using the Baermann funnel procedure, and total number of nematodes was counted under a microscope. Community composition and diversity of soil free-living nematodes were analyzed using 18S rDNA sequences. Results indicated that a total of 67 groups, including 64 species, 2 genera and 1 family were identified. Feeding behavior of 58 species were identified as follows: 15 bacteria-feeding species, 12 fungi-feeding species, 10 plant-parasite species, 5 omnivorous-predator species, 8 animal-parasite species, 5 invertebrate-parasite species and 3 non-free-living nematodes, known as marine species. Moreover, soil free-living nematodes were found to be affected by sampling locations and plant gender, and community composition and density of these nematodes were strongly influenced by soil OM content. Result confirmed that spatial location and plant cover were main factors influencing the diversity of soil free-living nematodes. Moreover, molecular tools were found to be very useful in defining the richness of soil non-free-living nematodes. In conclusion, the results elucidated the importance of biotic variables in determining the composition and abundance of soil free-living nematodes in the Namib Desert, Namibia.