Application of crop residues and biochar have been demonstrated to improve soil biological and chemical properties in agroecosystems. However, the integrated effect of organic amendments and hydrological cycles on soil health indicators are not well understood. In this study, we quantified the impact of hemp residue (HR), hemp biochar (HB), and hardwood biochar (HA) on five hydrolytic enzymes, soil microbial phospholipid (PLFA) community structure, pH, permanganate oxidizable carbon (POXC) soil organic carbon (SOC), and total nitrogen (TN). We compared two soil types, Piedmont and Coastal Plain soils of North Carolina, under (i) a 30-d moisture cycle maintained at 60% water-filled pore space (WFPS) (D-W1), followed by (ii) a 7-day alternate dry-wet cycle for 42 days (D-W2), or (iii) maintained at 60% WFPS for 42 days (D-W3) during an aerobic laboratory incubation. Results showed that HR and HB significantly increased the geometric mean enzyme activity by 1-2-fold in the Piedmont soil under the three moisture cycles and about 1.5-fold under D-W in the Coastal soil. In the presence of HA, the measured soil enzyme activities were significantly lower than control under the moisture cycles in both soil types. The shift in microbial community structure was distinct in the Coastal soil but not in the Piedmont soil. Under D-W2, HR and HB significantly increased POXC (600–700 mg POXC kg-1 soil) in the Coastal soil but not in the Piedmont soil while HA increased nitrate (8 mg kg-1) retention in the Coastal soil. The differences in amendment effect on pH SOC, TN, POXC, and nitrate were less distinct in the fine-textured Piedmont soil than the coarse-textured Coastal soil. Overall, the results indicate that, unlike HA, HR and HB will have beneficial effects on soil health and productivity, therefore potentially improving soil’s resilience to changing climate.