Shields diagram is used as the main source to determine the incipient motion of sediment. However, incipient sediment motion in the Shields diagram was developed based on non-cohesive sediment where the parameter influencing the motion of cohesive and non-cohesive sediments are different. Therefore, this study attempts to investigate the incipient sediment motion for biological extracellular polymeric substance (EPS) influenced by a cohesive sediment mixture. Percentage of silt with a median grain size of 28 μm (which acts as the cohesive material) and fine sand with of 150 μm were varied as 10:90, 20:80, 40:60, and 50:50, which formed the cohesive sediment mixture. Two different concentrations of EPS 0.02% (which denotes as low concentration) and 0.1% (as high concentration) were well mixed with the sediment mixture. This study utilised Xanthan gum, which acts as a substitution for EPS. The experiments were conducted in a laboratory flume and the threshold criterion for sediment motion was obtained through observation. The critical Shields parameter was calculated using the critical shear velocity and root-mean-square horizontal velocity (representing turbulent fluctuations) when few of the particles on the bed was observed to move. Obvious finding from this study is the presence of EPS clearly influence the threshold criteria based on the higher value obtained at sediment mixture with 0.1% compared to the values found for the 0.02% EPS sediment mixture. The values of the critical Shields parameter were monotonously increased as the percentage of silt in the sediment mixture increases. The presence of silt in the sediment mixture increases the sediment stability signifying more hydrodynamic forces are required for the particles to be entrained. The critical Shields parameter obtained based on the critical shear velocity and turbulent fluctuations posed similar trend as described in the well-established Shields curve indicating that the velocity scale used to describe the incipient sediment motion is not a decisive factor.