This study examined the saprophytic fungus Clonostachys rosea TNAU (Tamil Nadu Agricultural University) CR04, noted for its strong antagonistic capabilities against several plant pathogens, as a prospective biological control agent in sustainable agriculture. Moreover, this study aimed to optimize the growth conditions of C. rosea by examining the interactive effects of temperature, pH and water activity (aw) on its mycelial development. To accomplish this objective, we employed response surface methodology (RSM), specifically using a Box Behnken design, which allowed for a systematic exploration of these three critical variables across 17 experimental trials. The analysis revealed that temperature and pH positively affected growth, whereas relatively high-water activity negatively affected growth. The ideal conditions identified were 30 °C, pH 6.5 and aw of 0.88, resulting in a maximum radial growth of 44.80 mm. Model validation showed a strong correlation between the predicted and actual
outcomes, with an R² value of 0.9901. This research underscores the necessity of optimizing environmental parameters to improve the efficacy of C. rosea in agricultural applications. Future studies should focus on validating these findings under field conditions and examining the influence of additional environmental variables on various C. rosea strains to enhance the formulation of biofungicides and promote sustainable pest and disease management.