Delivering charged antibiotics to the intervertebral disc is challenging because of the avascular, negatively charged extracellular matrix (ECM) of the tissue. The purpose of this study was to measure the apparent diffusion coefficient of two clinically relevant, charged antibiotics, vancomycin (positively charged) and oxacillin (negatively charged) in IVD. A one-dimensional steady state diffusion experiment was employed to measure the apparent diffusion coefficient of the two antibiotics in bovine coccygeal annulus fibrosus (AF) tissue. The averaged apparent diffusion coefficient for vancomycin under 20% compressive strain was 7.94 ± 2.00 × 10−12 m2/s (n = 10), while that of oxacillin was 2.26 ± 0.68 × 10−10 m2/s (n = 10). A student’s t-test showed that the diffusivity of vancomycin was significantly lower than that of oxacillin. This finding may be attributed to two factors: solute size and possible binding effects. Vancomycin is approximately 3 times larger in molecular weight than oxacillin, meaning that steric hindrance likely plays a role in the slower transport. Reversible binding between positive vancomycin and the negative ECM could also slow down the rate of diffusion. Therefore, more investigation is necessary to determine the specific relationship between net charge on antibiotic and diffusion coefficients in IVD. This study provides essential quantitative information regarding the transport rates of antibiotics in the IVD, which is critical in using computational modeling to design effective strategies to treat disc infection.