The CDC biofilm reactor is a robust culture system with high reproducibility in which biofilms can be grown for a wide variety of analyses. Multiple material types are available as growth substrates, yet data from biofilms grown on biologically relevant materials is scarce, particularly for antibiotic efficacy against differentially supported biofilms. In this study, CDC reactor holders were modified to allow growth of biofilms on collagen, a biologically relevant substrate. Susceptibility to multiple antibiotics was compared between biofilms of varying species grown on collagen versus standard polycarbonate coupons. Data indicated that in 13/18 instances, biofilms on polycarbonate were more susceptible to antibiotics than those on collagen, suggesting that when grown on a complex substrate, biofilms may be more tolerant to antibiotics. These outcomes may influence the translatability of antibiotic susceptibility profiles that have been collected for biofilms on hard plastic materials. Data may also help to advance information on antibiotic susceptibility testing of biofilms grown on biologically relevant materials for future in vitro and in vivo applications.