The conversion of desert to farmland may change not only surface soils but also deeper soils due to massive irrigation and fertilization. However, limited information exists on the response of microorganisms throughout the soil profile to long-term crop development in desert regions. The shifts in the microbial community were investigated in relation to soil depth (0–2 m) and years of reclamation, as well as their relationship to soil organic carbon (SOC) dynamics in an irrigated arid area of northwest China. Oasis farmlands cultivated for 3, 5, 10, 20, 50, and > 100 years were compared with adjacent deserts. Total phospholipid fatty acids (PLFAs), bacterial PLFAs, and microbial biomass and activity within the whole profile increased from the beginning of farming and peaked at 20 years post-reclamation. Fungal and actinomycete PLFAs decreased in the beginning of reclamation but increased after 10 years and peaked at 20 years. Microbial community composition was dynamic within the first 10 years of reclamation and tended to become stable thereafter. During oasis formation, the carbon to nitrogen ratio of microbial biomass decreased from 9.0–10.5 to 7.2–8.0; the fungi to bacteria ratio decreased from 0.3–0.5 to 0.2–0.3, and the gram-positive to gram-negative bacteria ratio (GP:GN) increased from 0.3–0.4 to 0.4–0.6. Increases in microbial abundance and shifts in community structure were positively correlated with SOC accumulation. Redundancy analysis indicated that microbial changes were closely associated with increased soil moisture and nutrients and decreased salinity and pH. We believe the predominant factor driving microbial community shifts and soil carbon accumulation is the increased input of plant residue due to enhanced water and nitrogen availability after desert reclamation. This study emphasizes the significance of the microbial changes and the close association between increased GP:GN and SOC accumulation during oasis formation, especially in deeper soils (0.6–2.0 m).