<p>The health benefits of menaquinone-7 (MK-7) are well-established, and its production through fermentation techniques is widespread. Our team developed an innovative biofilm reactor utilizing <i>Bacillus subtilis natto</i> cells to foster biofilm growth on plastic composite supports to produce MK-7. The scalability of this biofilm reactor from a 2-L benchtop scale in our laboratory and its potential for commercial applications pose significant unresolved questions. Therefore, the current research was aimed to scale up the biofilm reactor from bench scale (2-L) to the pilot scale (30-L) bioreactor. Three strategies were evaluated to understand their impact on MK-7 biosynthesis during bioreactor volume expansion: volumetric oxygen mass transfer coefficient (<i>k</i>_La), agitation power input per unit volume (P/V), and impeller tip velocity (<i>V</i>_tip). While k_La was successfully maintained during scaling, P/V and V_tip varied and were assessed for their influence on MK-7 production. After investigating these methods, it was found that the volumetric oxygen mass transfer coefficient (k_La) constant method proved to be the most effective one. The optimum MK-7 concentration achieved was 21.0 ± 1.0 mg/L, comparable to the highest MK-7 concentration of 20.6 ± 1.0 attained at the 2-L scale. This showcases the scalability of biofilm bioreactor technology and its promising potential for commercial production of MK-7. Furthermore, we explored the potential of fed-batch glucose addition to the base media in the biofilm reactor to enhance MK-7 concentration at the 30-L scale. Remarkably, results demonstrated that fed-batch strategy significantly increased MK-7 concentrations to 28.7 ± 0.3 mg/L, which made it almost 2.3-fold higher than levels produced in suspended-cell bioreactors. This finding highlights the potential of biofilm reactors as a promising replacement to the current static fermentation strategies for commercial production of MK-7.</p>