Background: The objectives of this study were to create a protocol for making antibiotic impregnated Plaster of Paris (AI-PoP) beads using penicillin, ampicillin, tetracycline, tulathromycin, and florfenicol, to determine the in vitro elution rates of those antibiotics in the beads, and to quantify the in vitro inhibition of E. coli and T. pyogenes by the bead eluent in a proof-of-concept study using flow cytometry. The AI-PoP beads were made using Plaster of Paris powder, antibiotic, and tap water, cured for 24 hours, sterilized by ethylene oxide, and stored up to 5 months before testing. For each antibiotic, twenty beads were combined with bovine serum in sterile tubes and incubated at 37 C on a rocker. Serum was replaced at intervals over the 14 day study period, and antibiotic concentrations were determined by high pressure liquid chromatography with mass spectrometry. Separately, the growth of E. coli and T. pyogenes in eluent from 10 beads for each antibiotic was quantified by flow cytometry. Results: Antibiotic was detected in AI-PoP bead eluent for 14 days for all but the ampicillin beads, for which antibiotic was detected for 8 days. The concentration of antibiotic in eluent was greater than five times the minimum inhibitory concentration (MIC) of tested bacteria for the entire study period for penicillin, tetracycline, and florfenicol. The concentration of ampicillin remained greater than five times the MIC of E. coli for 4 days and T. pyogenes for 5 days. The concentration of tulathromycin remained greater than five times the MIC of E. coli for 5 days and T. pyogenes for the entire study period. The CFU/ml of live E. coli and T. pyogenes was reduced over a 72-hour period by 1-3 log 10 CFU, with the exception of tetracycline, which reduced CFU/ml of T. pyogenes by less than log 10 CFU. Conclusions: AI-PoP beads containing penicillin, tetracycline, tulathromycin and florfenicol elute antibiotic well above the MIC of tested bacteria for the two week study period and provide adequate local bacterial growth inhibition to reduce bacterial growth in vitro . These antibiotics show promise for delivery in joint and wound infections.