Studies that explore the transport and retention behavior of colloidal-sized microplastic (MP) with focusing on the governing mechanisms for their attachment and detachment process using colloidal-atomic force microscopy (C-AFM) were still limited. In the present study, multiscale investigations ranging from pore-scale column test to microscale visualization and eventually to nanoscale interfacial and adhesive force measurement were conducted. Pore- and microscale tests were conducted at various flow velocity and over a broad range of IS values and found that IS and flow velocity could synergically impact the deposition of MPs during filtration, in particular under unfavorable condition at small flow velocity. The net difference between the highest and lowest deposition conditions became smaller while flow velocity was decreasing in porous media. However, the net difference between the high and low IS conditions in parallel plate chamber were not sensitive to the change of flow velocity. The measurement from C-AFM suggested that not only the interfacial force but also the adhesive forces changed while MP was approaching/retracting to the collector surface. Information related to the magnitude, location, and occurrence of interfacial/adhesive forces were analyzed. Comparisons of the interaction energy determined from the measured force and ones derived from surface energy components using DLVO theory were conducted to explain the synergies of IS and flow velocity on pathogenic size MPs transport and deposition during filtration.