Land-use change and the conversion of land into croplands is one of the main drivers of soil degradation and the loss of soil organic carbon. Few studies have evaluated the impact of the expansion of the agricultural frontier on soil microbial communities in the Gran Chaco region in South America; therefore, the objective of this study was to explore how soil microbial communities respond to land-use changes in a semiarid region of Argentina. The land uses selected were total and selective clearing with livestock (TC-livestock and SC-livestock), total clearing with agriculture (TC-agriculture), and an undisturbed site (R-Chancaní). Our results suggest that microbial abundances were strongly influenced by seasonal changes. In both seasons, the lowest values of total microbial biomass were observed in TC-agriculture and TC-livestock.In the wet season, TC-agriculture had the lowest fungal abundance, and both total clearing sites (TC-livestock and TC-agriculture) had higher bacterial abundance than the other sites. Our data also showed that the Shannon and richness indices were highest at the farming sites, indicating a similar tendency in the diversity indexes in both dry and wet seasons. According to Pearson correlation analysis, humic substance (HS) content was a key driver influencing soil microbial communities, demonstrating that high HS content may significantly increase bacteria, fungi, and total phospholipid fatty acids. Microbial composition highly depended on seasonal changes. Overall, our results suggest that land-use change altered microbial community structure and bacterial diversity, and that HS content played a crucial role in driving changes in soil microbial composition.