Manure impacts labile pools of soil organic carbon (SOC) and nitrogen (N) which can influence soil microbial composition (MCC) and enzyme activities, and hence soil health. The present study was conducted to investigate the impacts of long-term dairy manure and inorganic fertilizers (INF) on soil carbon (C) as well as nitrogen (N) fractions, enzyme activities, and microbial community structure in different time horizons at planting (P), one month after planting (1MAP), and after harvesting (H) under corn (Zea mays L.)-soybean (Glycine max L.) rotation. Study treatments included three manure application rates (low, phosphorus-based recommended rate; medium, nitrogen-based recommended rate; and high, the double rate of medium nitrogen based recommended rate), two INF rates (medium only nitrogen additions; and high nitrogen, phosphorus, potassium, zinc, and sulfur additions) and a control (no application of manure and/or inorganic fertilizer). In comparison to the INF, the dairy manure not only significantly increased chemical fractions of C and N but also impacted the enzyme activities. Average urease activity after manure was applied was shown to be 26.8% higher than it was with INF applied at planting. The β-Glucosidase activity was 6 and 14% higher with manure than it was with INF at 1MAP and harvesting, respectively. The cold-water extractable nitrogen (CWEN) was enhanced with high manure rate at all timings of sampling compared to the high fertilizer rate (53%), and CK (90%). Principal component analysis indicated that MCC under manure differed from those under the INF treatments. The total bacteria/total fungi ratio at planting was increased with the INF compared to the manure addition. Pearson's correlation analysis showed that CWEC, CWEN, and enzyme activities especially β-Glucosidase activity were the key determinants of MCC. Data from this study showed that, compared to inorganic fertilizers, manure can be beneficial in enhancing soil health indicators.