This study examined biofilm thickness, density, and microbial composition in a full-scale MABR treating municipal wastewater, focusing on their spatial and operational variability. The MABR cassette arrangement created a thickness gradient, with biofilms in the front cassettes more than twice as thick as those at the back. Lower scouring intensity due to reduced airflow resulted in thicker biofilms. Microbial communities varied longitudinally and by operational phase, with thicker biofilms having a higher relative abundance of anaerobic microorganisms, such as fermenters and sulfur reducers, and fewer aerobic nitrifiers. Nitrosomonas were the main ammonia oxidizers, while Nitrospira and Ca. Nitrotoga dominated as nitrite oxidizers. The 16S RNA gene profiles showed strong correlations with biofilm thickness (R