Previous microplastic research under laboratory conditions has focused on microplastics that are homogeneously mixed into test media, in order to maximize test reproducibility and uniform bio-accessibility. Here we specifically focused on testing the idea that microplastics in soil could affect adjacent soil layers not containing microplastic themselves. We included two different microplastics (low-density polyethylene films and polyacrylonitrile fibers) and carried out a soil column test consisting of three different vertical layers (0–3 cm, top, control soil; 3–6 cm, middle, microplastic-containing soil; 6–9 cm, bottom, control soil). Our study shows that microplastic-containing soil layers can act as an anthropogenic barrier in the soil column, interrupting the vertical water flow. These changes directly affected the water content of adjacent layers, and changes in the proportion of soil aggregate sizes occurred for each depth of the soil columns. We also observed that these physical changes trigger changes in soil respiration, but do not translate to effects on enzyme activities. These results imply that the soil environment in non-contaminated parts of the soil can be altered by microplastic contamination in adjacent layers, as might occur for example during ploughing on agricultural fields. More generally, our results highlight the need to further examine effects of microplastic in experiments that do not treat this kind of pollution as uniformly distributed.