Laboratory grown biofilms are used to simulate bacterial growth in diverse environmental conditions and screen the effectiveness of anti-biofilm therapies. Recently, we developed a glass bead biofilm reactor that utilizes low broth volume to provide high-throughput biofilm growth for testing and translation across the research continuum (e.g., benchtop assays to preclinical models). Bioburden per mm2 surface area of Staphylococcus aureus and Pseudomonas aeruginosa biofilms were comparable on beads and CDC Biofilm Reactor® coupons. In this study, we hypothesized that biofilms grown on beads would be more susceptible to ertapenem, moxifloxacin, and tobramycin than those grown on coupons. Results indicated a significant reduction in S. aureus bioburden on glass beads compared to glass coupons following treatment with ertapenem (p = 0.005) and tobramycin (p = 0.014). P. aeruginosa biofilms had smaller differences in antibiotic response between the two systems. There was a significantly greater reduction in bead P. aeruginosa biofilm than coupon when treated with tobramycin (p = 0.035). This work offered insight into how the bead biofilm reactor could be used as a tool for antibiotic screening and translation across the continuum of in vitro to in vivo systems that support development of antimicrobial technology.