In unsaturated soil columns, the boundary condition imposed at the column outlet may cause experimental artifacts. Our objective was to study in situ colloid mobilization during transient, unsaturated flow as affected by the boundary condition at the column outflow. We conducted colloid mobilization experiments by infiltrating unsaturated sandy sediment columns under different bottom boundary conditions: a seepage and a suction control. The mechanisms of colloid mobilization were investigated using force calculations (adhesive and interfacial forces), complemented with flotation experiments, where colloids in the bulk fluid and at the liquid–gas interface were measured separately. More colloids were mobilized under seepage than under suction-controlled boundary conditions. The shape of the colloid breakthrough curves also differed: for the seepage boundary, the maximum of the colloid concentration occurred at the beginning of the column outflow, but for the suction-controlled boundary, colloid concentrations in the outflow increased gradually before reaching a maximum. Colloid mobilization increased with flow rate and decreased with ionic strength for both boundary conditions; however, colloids were mobilized even at ionic strength exceeding the critical coagulation concentration (CCC). Flotation experiments showed that colloids were located both in the bulk fluid and at the liquid–gas interface at electrolyte concentrations less than the CCC, but only at the liquid–gas interface when the CCC was exceeded. Theoretical considerations confirm that interfacial forces at the liquid–gas interface exceeded adhesive forces at all ionic strengths. Both experiments and theory show that the liquid–gas interface had a dominant effect on colloid mobilization.