Recent evaluations have demonstrated the ability of the bacteria Rhodococcus rhodochrous DAP 96253 to inhibit the growth of molds associated with plant and animal diseases as well as post-harvest loss of fruits, vegetables and grains. Pre-pilot-scale fermentations (20-30L) of Rhodococcus rhodochrous DAP 96253 were employed as a research tool with the goal of producing a practical biological agent for field-scale application for the management of white-nose syndrome (WNS) in bats and post-harvest fungal losses in several fruit varieties. Several key parameters within the bioreactor were evaluated for the potential to increase production efficiency as well as activity of the biocatalyst. These parameters included elapsed fermentation time, dissolved Oxygen, and carbohydrate concentration of which increased carbohydrate concentration at the time of harvest was shown to have a negative impact on the catalyst activity. In addition, process improvements including utilization of a liquid inoculum, an autoinduction feed strategy, and increased glucose concentration in the feed medium increased fermentation yields to 100-150g/L, while the biocatalyst efficiency was increased from previous work. To increase production efficiency, a multi-bioreactor scheme was developed that used a seed bioreactor and subsequent production tank, which doubled run yields per production cycle. Amidase, cyanidase, urease, and alkene-monoxygenase activity were monitored throughout the study as potential indicators for the multi-faceted mechanism of fungal antagonism. Of these amidase, cyanidase, and urease were demonstrated to be more elevated in cells that showed antifungal activity than those that did not. This study represents the first example of a reproducible pre-pilot plant-scale biomanufacturing process for a contact-independent biological control agent for established and emerging fungal pathogens of plants and animals, and facilitates large-scale production for broad application.