Optimizing fermentation conditions is crucial in maximizing the production of bioactive metabolites and improving the consistency and efficacy of microbial biocontrol agents. This study investigates the influence of specific carbon and nitrogen sources on the growth, cyclolipopeptide (cLP) production, and antifungal activity of Bacillus amyloliquefaciens strain VFS2 against Fusarium equiseti. Batch fermentations were conducted in four media types (LB, LA, GA, and PM) across five incubation periods (0, 8, 24, 48, and 72 hours). A one-factor-at-a-time approach followed by optimization using Response Surface Methodology (RSM) revealed that the highest antifungal activity (up to 95%) was achieved in PM and LB media. HPLC profiling showed that the composition of the culture media significantly influenced the production of key cLPs, including isoforms of iturins, fengycins, and surfactins. Pearson correlation analysis confirmed a strong, significant association (p < 0.001) between specific cLPs isoforms and the antifungal activity of VFS2, with media-dependent variability. These results demonstrate the potential of integrated metabolic profiling and RSM-based optimization to enhance the biocontrol performance of B. amyloliquefaciens VFS2 under tailored fermentation conditions.