The rheological properties of membrane biofouling layers developing in Membrane Bioreactors (MBR) have not been measured directly so far, although they are essential for understanding biofouling and for determining conditions to mitigate it. The development of a novel method is reported herein, which permits accurate rheological measurements on membrane biofouling layers, using specimens withdrawn from an operating laboratory-scale MBR. A specially designed test-section (with a pair of flat membrane specimens) allows implementation of realistic operating modes, including automatic periodic back-washing. The membrane specimens, with a biofouling layer representative of a well-controlled MBR operating history, are examined in an advanced parallel-plate rheometer, where special care is taken to prevent ex-situ biofouling layer changes. Measurements performed in oscillatory and steady shearing modes provide valuable information on biofouling layer rheological characteristics, confirming that these layers are typical viscoelastic materials with yield stress exhibiting thixotropic, shear-thinning behavior. Rheological properties of the activated sludge liquor are also assessed. The rheological properties of activated sludge and of biofouling layers display qualitative similarities, although the elastic character of the latter is much more pronounced. The new technique is most useful for studying biofouling layers as a function of the main MBR process parameters. Improvements of the technique for such applications are discussed.