Growth of bacterial cells within a biofilm complicate the treatment of infections. Therefore, in the present study biofilm eradication efficacy of (ceftriaxone and sulbactam plus EDTA; CSE1034) was compared with ceftriaxone alone, ceftriaxone plus EDTA and ceftriaxone plus sulbactam against biofilms of ESBL producing Escherichia coli, Klebsiella pneumoniae and Salmonella typhi. Susceptibility testing of each drug was performed on planktonic and biofilm cells in non ESBL producing and ESBL producing strains according to the recommendations of clinical and laboratory standards institutes guidelines. CSE1034 inhibited the growth of planktonic cells of non ESBL producing strains with minimum inhibitory concentration (MIC) from 0.25 to 1.0 μg/ml; the minimum biofilm eradication concentration (MBEC) values ranged from 8 to 32 μg/ml where as ESBL producing strains MIC values were 2 to 4 times higher and corresponding MBEC values were higher by 4 to 8 times. When biofilms of ESBL producing organisms were treated with the half MBEC of drugs, CSE1034 decreased 3 log of bacteria present in biofilm when compared with ceftriaxone, ceftriaxone plus EDTA and ceftriaxone plus sulbactam. In conclusion, combination of CSE1034 acts synergistically and reduces the MIC and MBEC values, significantly. One dimensional polyacrlamide gel elctrophoresis of extracellular proteins revealed distinct difference in protein expression of the group treated with CSE1034. Hence, CSE1034 at low concentration showed greater efficacy in the eradication of biofilm as compared to other two drugs and could be one of the best choices to eradicate the biofilm infections caused by these organisms as compared to other drugs.