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Aims:  The aim of this study was to isolate arsenic‐resistant bacteria from contaminated sediment of the Orbetello Lagoon, Italy, to characterize isolates for As(III), As(V), heavy metals resistance, and from the phylogenetic point of view. Methods and Results:  Enrichment cultures were carried out in the presence of 6·75 mmol l−1 of As(III), allowing isolation of ten bacterial strains. Four isolates, ORAs1, ORAs2, ORAs5 and ORAs6, showed minimum inhibitory concentration values equal or superior to 16·68 mmol l−1 and 133·47 mmol l−1 in the presence of As(III) and As(V), respectively. Isolate ORAs2 showed values of 1·8 mmol l−1 in the presence of Cd(II) and 7·7 mmol l−1 of Zn(II), and isolate ORAs1 pointed out a value of 8·0 mmol l−1 in the presence of Cu(II). Analysis of 16S rRNA gene sequences revealed that they can be grouped in the three genera Aeromonas, Bacillus and Pseudomonas. Phylogenetic analysis of the four more arsenic‐resistant strains was also performed. Conclusion:  Isolates are highly resistant to both As(III) and As(V) and they could represent good candidates for bioremediation processes of native polluted sediments. Significance and Impact of the Study:  This study provides original results on levels of resistance to arsenic and to assigning genera of bacterial strains isolated from arsenic‐polluted sediments.