In the pursuit of new antifungal compounds, five coproduced lipopeptide variants (AF(1) to AF(5)) from wild-type Bacillus subtilis RLID 12.1 were identified in our previous study. Out of five, AF(4) was identified as a novel lead molecule belonging to the bacillomycin family showing less cytotoxicity at its respective minimum inhibitory concentrations (MIC) evaluated against 81 strains of Candida and Cryptococcus species (including clinical isolates); besides this, AF(4) purified in the present study exhibited encouraging MIC values against 10 clinical mycelial fungi. Aiming for a selective production augmentation of AF(4) lipopeptide variant, a new fermentation media comprising malt extract (1.01%), dextrose (0.55%), peptone (1.79%), MnSO4 (2mM), and NaCl (0.5%) was formulated. Maximum production of 954.8 +/- 10.8mg/L was achieved with 44% selectivity at 30 degrees C compared to unoptimized conditions (186.4 +/- 6.1mg/L). Use of calcium alginate beads in the formulated media during the onset of lipopeptide production resulted in an augmentation in the selectivity of the most efficacious AF(4) variant to about 72% presumably due to attenuation of other coproduced lipopeptide variants AF(1) and AF(2). Difference in yield of lipopeptides varied with bead size, bead preparation ratios, and sodium alginate concentrations. Use of Ca-alginate beads in the upstream production process of the lead AF(4) variant may be considered as a novel strategy to address the potential challenge that may arise during the scale-up and downstream processing steps. Another significant finding derived from the study is that the proportion of bacillomycin variants of B. subtilis RLID 12.1 could be controlled by temperature and metal ions under static and shaking conditions.