Using moderate populations of filaments in the biomass of Membrane Bio-Reactors (MBRs) is a biological anti-fouling method which has been increasingly applied over the last few years. This study aims to quantify the effect of COD to TN ratio, Dissolved Oxygen (DO) concentration and temperature on filaments’ population and Trans-Membrane Pressure (TMP) in a pilot-scale MBR, with a view to reducing membrane fouling. The novelty of the present work concerns the development of a mathematical equation that correlates fouling rate (dTMP/dt) with the population of filamentous microorganisms, assessed by the Filament Index (FI), and with the concentration of the carbohydrate fraction of Soluble Microbial Products (SMPc). Apart from TMP and SMPc, other fouling-related biomass characteristics, such as sludge filterability and settleability, were also examined. It was shown that at high COD to TN ratio (10:1), low DO concentration in the filaments’ tank (0.5 ± 0.3 mg/L) and high temperature (24−30 °C), a moderate population of filaments is developed (FI = 1−2), which delays the TMP rise. Under these conditions, sludge filterability and settleability were also enhanced. Finally, TMP data analysis showed that the fouling rate is affected by FI and SMPc concentration mainly in the long-term fouling stage and increases exponentially with their increase.