Harvester ants (Messor spp.) function as an essential link between aboveground resources and belowground biota such as the microbial community. We examine changes in soil microbial biomass and functional diversity resulting from harvester ant (Messor spp.) activity in the Negev Desert, Israel. Abiotic and biotic soil parameters were recorded during two seasons – wet and dry – also representing food availability periods for the ants (low and high seed availability, respectively). Soil samples were collected monthly from the 0- to 10- and 10- to 20-cm soil layers: (1) near the nest entrance, (2) under chaff piles, and (3) at a 2-m radius from the nest entrance (control). Harvester ant activity increased the percentage of organic matter, total soluble nitrogen, and microbial activity in nest-modified soils in comparison to the control soils. Higher CO2 evolution was recorded in the low-seed season in ant nest soils than in the control soils. During the high-seed season, higher carbon dioxide evolution was recorded only at the nest entrance locations. There were no differences in microbial biomass between the low- and high-seed seasons, but highest microbial biomass was found under chaff in low-seed season and in nest soils in high-seed season. Microbial function diversity was higher in nest-modified soils than in the control soils. This study suggests that the effect of harvester ant nests on soil fertility is due to increased microbial biomass and microbial activity in ant nest-modified soils.