Recent studies identified and established a platform of polymer surface modifications allowing the attachment of penicillin (PEN) to expanded poly(tetrafluoroethylene) (ePTFE) surfaces. The effectiveness of this approach was accomplished by creating surfaces with chemically attached PEN that prevent the proliferation of microbes. In this study, quantitative assessments of PEN effectiveness attached to ePTFE were conducted. Using variable-angle attenuated total reflectance (ATR-FTIR) spectroscopy, the volume coficentration changes of PEN were determined as a function of depth from the ePTFE surface. At depths ranging from 0.2 to 1.2 mu m from the surface, PEN concentration levels decrease from 8.85 to 3.33,mu g/M-3. Assessment of concentration levels of the colony forming units (CFUs) of Staphylococcus aureus bacteria as a function of contact time with the penicillin-polyethylene glycol spacer separated by maleic anhydride ePTFE (PEN-PEG-MA-ePTFE) surfaces showed profound effectiveness of PEN in preventing microbial proliferation. Hydrolytic stability of PEN-PEG-MA-ePTFE surfaces revealed that even with a 32% loss of PEN due to the cleavage of the ester linkages between PEN and PEG spacer, antimicrobial activity is still maintained.