A novel quorum quenching (QQ) strain, Acinetobacter guillouiae ST01, was isolated from a full-scale membrane bioreactor (MBR) and characterized for its QQ activities. Batch reactor studies at lab-scale showed that A. guillouiae ST01 exhibited higher QQ activity against acyl homoserine lactones (AHLs) with an oxo group compared to those without an oxo group. The organism was then inoculated (10%) in an MBR (Q-MBR) treating sewage over 48 days and was found to reduce quorum sensing (QS) activity by reducing AHL concentrations in the sludge and the biofilm of the Q-MBR. The concentration of polysaccharides was reduced up to 30% in both the biofilm and sludge relative to the control, whereas protein concentrations were reduced by 40% and 47% in the sludge and biofilm, respectively. The Q-MBR fouling rates were halved. These results indicate that A. guillouiae ST01 is a promising strain for biofouling reduction in MBR treating real wastewater.