Arsenite-oxidizing bacteria (AOB) play a key role in the biogeochemical cycle of arsenic in the environment, and are used for the bioremediation of As contaminated groundwater; however, it is not yet known about how arsenic affects biofilm formations of AOB, and how biofilm formations affect bacterial arsenite-oxidizing activities. To address these issues, we isolated seven novel AOB strains from the arsenic-contaminated soils. They can completely oxidize 1.0 mM As(III) in 22–60 h. Their arsenite oxidase sequences show 43–99% identities to those of other known AOB. Strains Cug1, Cug2, Cug3, Cug4, and Cug6 are able to form biofilms with thickness of 15–95 µm, whereas Cug8 and Cug9 cannot form biofilms. It is interesting to see that arsenite inhibited the biofilm formations of heterotrophic AOB strains, but promoted the biofilm formations of autotrophic strains in a concentration-dependent manner. The arsenite-oxidizing rates of Cug1 and Cug4 biofilms are 31.6% and 27.6% lower than those of their suspension cultures, whereas the biofilm activities of other strains are similar to those of their suspension cultures. The biofilm formation significantly promoted the bacterial resistance to arsenic. This work is the first report on the complex correlations among environmental arsenic, bacterial biofilm formations and bacterial arsenite-oxidizing activities. The data highlight the diverse lifestyle of different AOB under arsenic stress, and provide essential knowledge for the screening of efficient AOB strains used for constructions of bioreactors. Display omitted •Seven novel As(III)-oxidizing bacterial strains were isolated.•As has differential effects on the biofilm formations of the strains.•Biofilm formations either inhibit or slightly affect the activities of the strains.•Biofilm formation significantly promotes bacterial resistance to As.•The findings in this work are helpful for bioreactor constructions.