Bacterial multidrug resistance is a major cause of clinical failures in treating infections. In this study, Escherichia coli and Pseudomonas aeruginosa were used as model systems to explore the genetic basis of multidrug resistance. The random mutant libraries were constructed in E. coli EC100 and P. aeruginosa PAO1 using transposon mutagenesis. The libraries were screened to identify the mutants with enhance/reduced resistance to antibiotics. Out of the 4000 E. coli mutants screened up to date, 6 mutants were found to be more sensitive to chloramphenicol and 7 were more resistant compared to the wild-type EC100. Thirty-three drug sensitive mutants and 1 drug resistant mutant were identified based on 3000 P. aeruginosa mutants that have been screened. Thirty-four genes responsible for these phenotypic changes were identified by inverse PCR and DNA sequencing. The functions of these genes and their roles in biofilm multidrug resistance were explored. The gene rob of E. coli was found to be important for drug resistance in biofilms.