Several studies have suggested the existence of a close relationship between antibiotic-resistant phenotypes and resistance to other toxic compounds such as heavy metals, which involve co-resistance or cross-resistance mechanisms. A metagenomic library was previously constructed in Escherichia coli with DNA extracted from the bacterial community inhabiting an acid mine drainage (AMD) site highly contaminated with heavy metals. Here, we conducted a search for genes involved in antibiotic resistance using this previously constructed library. In particular, resistance to antibiotics was observed among five clones carrying four different loci originating from CARN5 and CARN2, two genomes reconstructed from the metagenomic data. Among the three CARN2 loci, two carry genes homologous to those previously proposed to be involved in antibiotic resistance. The third CARN2 locus carries a gene encoding a membrane transporter with an unknown function and was found to confer bacterial resistance to rifampicin, gentamycin, and kanamycin. The genome of Thiomonas delicata DSM 16361 and Thiomonas sp. X19 were sequenced in this study. Homologs of genes carried on these three CARN2 loci were found in these genomes, two of these loci were found in genomic islands. Together, these findings confirm that AMD environments contaminated with several toxic metals also constitute habitats for bacteria that function as reservoirs for antibiotic resistance genes.