Fungal infections represent an enormous load on the public especially with the development of resistance to the most currently used antifungal drugs in practice. In the present work, a bacterial isolate coded A3 was recovered from soil and was shown to express antagonistic activity against Candida (C.) albicans ATCC 10231. This isolate was identified as Bacillus (B.) subtilis subsp. spizizenii isolate CCASU 2021-4 using 16S ribosomal RNA sequencing. D-optimal design from response surface methodology (RSM) was used to optimize the environmental variables affecting the antifungal activity of the respective isolate. The optimum conditions were a temperature of 30 °C, a pH of 8, and an inoculum size of 5 x 10 7 cfu/mL, resulting in an enhancement in the antifungal activity by 1.2 fold. This is the first report, to the best of our knowledge, on an antifungal activity from B. subtilis subsp. spizizenii culture broth against human fungal pathogens along with its optimization through RSM.