Acidophilic bacterium, Acidiphilium symbioticum H8, is resistant to high levels of several heavy metals, hydrophobic agents, and organic solvents. The approximately 9.6 kb plasmid pASH8, was purified, digested with HindIII, and sub-cloned in pUC19 at the respective site. Three different fragment size clones were achieved. The clones were completely sequenced and analyzed. The first clone encodes for a single putative open reading frame (ORF), which showed significant homology to several rusticyaninA1 proteins. The second clone encodes for a 43-kDa protein, which has conserved domain homology with several outer envelop TolC proteins. The clone with pASH8 tolC gene can functionally complement an Escherichia coli tolC mutant strain, making it resistant to several toxic hydrophobic agents, earlier for which it was sensitive. The tolC gene was found to be essential for imparting resistance to the clone toward these toxic hydrophobic agents. The third clone encodes for a putative 318-aa AcrA (acriflavine resistance protein A) protein and the clone was resistance to plasmid curing dye acriflavine. The clone also has a truncated ORF, which showed significant homology to cation-efflux pump AcrB. This study is the first to report a multi-drug efflux system to be encoded on a plasmid of any Acidiphilium strain.