Efforts to address economic and environmental concerns surrounding nitrogen (N) have motivated attempts to improve estimates of plant-available N in soil. Several soil health indicators, including CO2 burst, permanganate oxidizable carbon (C) (POXC), and autoclaved-citrate extractable (ACE) soil protein, assess labile C and N, and therefore may help to estimate soil N mineralization in long-term cover cropping systems (>3 years). This study evaluated the relationship of CO2 burst, POXC, ACE-soil protein, and pre-sidedress nitrate test (PSNT) with agronomic optimum N rate (AONR) in corn (Zea mays L.). The study also looked at relationship between other soil test and corn yield parameters, relative yield (RY) and yield without N sidedress at 25 long-term cover crop sites across Virginia. Results showed relatively weak correlations between AONR and CO2 burst, POXC, ACE-soil protein, and NO3-N (r = 0.00 to -0.48), which indicates that these soil health tests may not reliably predict soil N availability and corn yield. Corn yield with zero-sidedress N rate had a negative relationship with cover crop C:N ratio (r = -0.66) and a positive relationship with cover crop N content (r = 0.59), and NO3-N at pre-planting (r = 0.54) and sidedress (PSNT) (r = 0.63). The PSNT showed a better relationship (r = 0.65) compared to 72-h CO2 burst (r = 0.48) with RY. Soil health indicators (CO2 burst, POXC and ACE-soil protein) resulted in a poor or no relationship with AONR. Our results indicate that the PSNT was a more reliable indicator of the sidedress N rate in corn.