The soil’s biological health is an important aspect in sustaining crop production in arable soils. The impact of the long-term application of graded doses of farmyard manure(FYM) and its integration with mineral nitrogen on soil biochemical properties was investigated in a pearl millet-wheat system. The study involved four levels of FYM (0, 10, 20 and 30 Mg ha-1) and two nitrogen levels (0 and 120 kg ha-1) and the effects were assessed soil nitrogen availability, microbial activity and nitrogen (N) cycling soil enzymes activities in two soil depths (0-15 and 15-30 cm). The results showed a significant increase (p< 0.05) in soil available nitrogen with addition of FYM and N and it ranged from 169-328 kg ha-1 in surface soil and 132-238 kg ha-1 in subsurface soil. The soil microbial biomass carbon (MBC) and nitrogen (MBN) content was significantly increased with increasing FYM and N levels, where MBC ranging from 162-1189 mg g-1 soil in 0-15 cm depth and 91-852 mg g-1 soil in 15-30 cm depth. The substrate-induced respiration (SIR) and soil microbial quotient (SMQ) was also reported with FYM and N application irrespective of soil depths. The soil urease activity was increased by 227% in surface soil and 323% in subsurface soil with increased FYM level from 0 to 30 Mg ha-1. The activity of arginine deaminase in soil ranged from 4.36-10.3 µg NH4+ g-1 soil hr-1and 2.60to 6.34 µg NH4+ g-1 soil hr-1in 0-15 cm and 15-30cmsoil layer, respectively. Thus, the study emphasizes the importance ofFYM and N fertilization for enhancing soil microbial biomass,activity and nitrogen availability in semiarid environment, all of which support better soil health and sustainable crop production.