The colonization of pozzolana by an As(III)- oxidizing bacterial consortium was monitored from the first hours of bacterial adhesion to 6 weeks of development under fed-batch conditions, using adapted ultrasonic dislodging and crystal-violet staining procedures to determine the biofilm adhering to the complex surfaces. The effect of temperature, arsenic concentration, and presence or absence of yeast extract (YE) on the amount of biofilm biomass and on the As(III)-oxidation were assessed to test the biofilm's resilience and optimize the colonization. Fed-batch cultures allow twice as much pozzolana colonization as that obtained under batch conditions. In addition, As(III) oxidation and the quantities of biomass under fed-batch culture conditions were the same at 14°C and 25°C. Whereas YE improves (+150%) bacterial adhesion during the first 2 h, its impact in the longer term appears to be less significant—biofilm formation in presence of YE after 5 weeks was no greater than biofilm formation in the absence of YE. Finally, YE involves a drastic (−70%) decrease of As(III) oxidation. Preliminary tests for drinking-water bioremediation revealed the ability of Chéni Arsenic Oxidizing 1 biofilms to remain and retain As(III) oxidation activity at low As(III) concentrations (50 μg l−1).