The genomes of several Acinetobacter species possess three distinct polysaccharide-producing operons [two poly-N-acetyl glucosamine (PNAG) and one K-locus]. Using a microfluidic device, an increased amount of polysaccharides and enhanced biofilm formation were observed following continuous exposure to H2O2 and removal of the H2O2-sensing key regulator, OxyR, in Acinetobacter oleivorans DR1 cells. Gene expression analysis revealed that genes located in PNAG1, but not those in PNAG2, were induced and that genes in the K-locus were expressed in the presence of H2O2. Interestingly, the expression of the K-locus gene was enhanced in the PNAG1 mutant and vice versa. The absence of either OxyR or PNAG1 resulted in enhanced biofilm formation, higher surface hydrophobicity, and increased motility, implying that K-locus-driven polysaccharide production in both the oxyR and PNAG1 deletion mutants may be related to these phenotypes. Both the oxyR and K-locus deletion mutants were more sensitive to H2O2 compared with the wildtype and PNAG1 mutant strains. Purified OxyR binds to the promoter regions of both polysaccharide operons with a higher affinity toward the K-locus promoter. Although oxidized OxyR could bind to both promoter regions, the addition of dithiothreitol further enhanced the binding efficiency of OxyR, suggesting that OxyR might function as a repressor for controlling these polysaccharide operons.