In the present study, we investigated the impact of treated wastewater (TWW) irrigation on the prevalence of antibiotic resistance genes (ARGs) in subsoil pore-water, a so-far under-appreciated matrix. We hypothesized that TWW irrigation increases ARG prevalence in subsoil pore-water. This hypothesis was tested using a multiphase approach, which consisted of sampling percolated subsoil pore-water from lysimeter-wells of a real-scale TWW-irrigated field, operated for commercial farming practices, and controlled, laboratory microcosms irrigated with freshwater or TWW. We monitored the abundance of six selected ARGs (sul1, blaOXA-58, tetM, qnrS, blaCTX-M-32 and blaTEM), the intI1 gene associated with mobile genetic elements and an indicator for anthropogenic pollution and bacterial abundance (16S rRNA gene) by qPCR. The bacterial load of subsoil pore water was independent of both, irrigation intensity in the field study and irrigation water type in the microcosms. Among the tested genes in the field study, sul1 and intI1 exhibited constantly higher relative abundances. Their abundance was further positively correlated with increasing irrigation intensity. Controlled microcosm experiments verified the observed field study results: the relative abundance of several genes, including sul1 and intI1, increased significantly when irrigating with TWW compared to freshwater irrigation. Overall, TWW irrigation promoted the spread of ARGs and intI1 in the subsoil pore-water, while the bacterial load was maintained. The combined results from the real-scale agricultural field and the controlled lab microcosms indicate that the dissemination of ARGs in various subsurface environments needs to be taken into account during TWW irrigation scenarios.