Viable cells of Escherichia coli were entrapped in agar gel layers to form artificial biofilm-like structures. Killing assays of immobilized organisms by latamoxef and tobramycin were performed under different oxygenation conditions of the culture medium and compared with free-cell experiments. Under moderate aeration, agar-entrapped bacteria displayed higher resistance to the two antibiotics than suspended cells. Slow-growing free-cell cultures were resistant to latamoxef but not to tobramycin. In anaerobic incubation conditions, suspended organisms were highly resistant to the two antibiotics. Sustained oxygenation enhanced tobramycin efficacy against free and immobilized cells. These results show that oxygen deficiency in the gel layer contributes to the enhanced antibiotic resistance of sessile-like cells.