A two-year study (harvest years 2019 and 2020) was conducted to investigate the effect of a commercially available biofertilizer, in combination with variable nitrogen (N) rate, on bread baking quality and agronomic traits in hard winter wheat grown in conventional (CONV) and organic (ORG) farming systems in Kentucky, USA. The hard red winter wheat cultivar ‘Vision 45’ was used with three N rates (44, 89.6 and 134.5 kg/ha as Low, Med and High, respectively) and three biofertilizer spray regimes (no spray, one spray and two sprays). All traits measured were significantly affected by the agricultural production system (CONV or ORG) and N rate, although trends in their interactions were inconsistent between years. In Y2, yield was greatest in treatments with high N rates and in the ORG system. Biofertilizer treatments had a negative to neutral effect on grain yield. Baking quality traits such as protein content, lactic acid solvent retention capacity and sedimentation value (SV) were consistently greater in the CONV system and increased with the higher N application rates. Similarly, biofertilizer application had no effect on predictive baking quality traits, except for SV in year 1 of the study, where it increased with two sprays. Loaf volume was consistently greater from wheat grown in CONV treatments. From these results, we conclude that further research is warranted to evaluate the potential for biofertilizers to enhance N uptake and affect bread baking quality or other end-use traits. Additional research may be especially useful in organic production systems where biologically based N fertilizers are utilized, and treatments were not negatively affected by biofertilizer applications. Such strategies may be needed to increase protein quantity and gluten quality to optimize winter wheat production for bread baking qualities in the southeastern USA.