Using minimal salts medium containing vitamins and pyruvate (MSVP) plus added manganese sulfate, a manganese (II) oxidizing bacterial strain was isolated from water samples of a biofiltration system treating borehole water in KwaZulu-Natal, South Africa. The nonmotile Gram-negative and oxidase-negative isolate was subsequently characterized microbiologically. Based on its morphological and physiological characteristics and on the analysis of its 16S rRNA gene sequence, the isolate was assigned to the genus Acinetobacter. Growth of the isolated strain in MSVP with added manganese sulfate gave rise to a drop in pH and a concomitant increase in oxidation–reduction potential, which was absent in controls, thus indicating manganese oxidation. The ability of Acinetobacter sp. strain LB1 to oxidize Mn (II) was further verified using the leucoberbelin blue dye assay as well as by energy dispersive X-ray analysis of crystals formed in the medium. In addition, a biofilm assay indicated that this isolate can attach to solid surfaces such as a biofilter matrix, thus confirming its potential for biotechnological applications in the area of water purification.