The management of agro-waste and its conversion into useable products using biotechnological applications in agriculture is garnering significant interest. Bacillus strains are commonly used in poultry cultures as probiotics or single-cell proteins. However, their high production costs and low biomass yields limit their use as microbial proteins in animal feeds.  Biomass production of Bacillus licheniformis and Bacillus subtilis is a growing area of interest in agriculture due to its nutritional benefits. This study evaluated various agro-waste culture media for biomass production of these probiotics, utilizing banana, pea, rice husks, and potato peels. The highest biomass production was achieved by Bacillus licheniformis (CCASU-2024-65) at 1.97 g/L with banana peel extract medium, while Bacillus subtilis (CCASU-2024-67) recorded high biomass production of 1.5 g/L with potato peel extract medium. The statistical Plackett-Burman Design was used to screen nutrient components and culture conditions for these probiotics. The maximum biomass production of Bacillus licheniformis was 2.7 g/l, achieved at run number 8, which had ideal conditions comprising an initial pH of 6, 50 g/L of banana peel extract, 50 g/L of pea peel extract, a 10% inoculation ratio, and a fermentation duration of 48 hours for biomass generation. Bacillus subtilis produced 3.52 g/l output at run number 12, which had output occurred at run number 12, which comprised 50 g/L potato peel extract and 50 g/L pea peel extract concentration, an initial pH of 7, a 10% inoculum size, and a fermentation duration of 48 hours.