Pseudomonas aeruginosa is an opportunistic pathogen that produces sessile communities known as biofilms that are highly resistant to antibiotic treatment. Limited information is available on the exact role of various components of the matrix in biofilm-associated antibiotic resistance. Here we show that the presence of extracellular polysaccharide reduced the extent of biofilm-associated antibiotic resistance for one class of antibiotics. Minimal bactericidal concentration (MBC) for planktonic and biofilm cells of P. aeruginosa PA14 was measured using a 96 well microtiter plate assay. The MBC of biofilm-grown ΔpelA mutant, which does not produce the Pel polysaccharide, was 4-fold higher for tobramycin and gentamicin, and unchanged for ΔbifA mutant, which overproduces Pel, when compared to the wild type. Biofilms of pelA mutants in two clinical isolates of P. aeruginosa showed 4- and 8-fold higher MBC for tobramycin as compared to wild type. There was no difference in the biofilm resistance of any of these strains when tested with fluoroquinolones. This work forms a basis for future studies revealing the mechanisms of biofilm-associated antibiotic resistance to aminoglycoside antibiotics by P. aeruginosa [Int Microbiol 2010; 13(4):207-212]