Antibiotic resistance (AR) is a global phenomenon with severe epidemiological ramifications. Anthropogenically impacted natural aquatic and terrestrial environments can serve as reservoirs of antibiotic resistance genes (ARG), which can be horizontally transferred to human-associated bacteria through water and food webs, and thus contribute to AR proliferation. Treated-wastewater (TWW) irrigation is becoming increasingly prevalent in arid regions of the world, due to growing demand and decline in freshwater supplies. The release of residual antibiotic compounds, AR bacteria, and ARGs from wastewater effluent may result in proliferation of AR in irrigated soil microcosms. The aim of this study was to assess the impact of TWW-irrigation on soil AR bacterial and ARG reservoirs. Tetracycline, erythromycin, sulfonamide, and ciprofloxacin resistance in soil was assessed using standard culture-based isolation methods and culture-independent molecular analysis using quantitative real-time PCR (qPCR). High levels of bacterial antibiotic resistance were detected in both freshwater- and TWW-irrigated soils. Nonetheless, in most of the soils analyzed, AR bacteria and ARG levels in TWW-irrigated soils were on the whole identical (or sometimes even lower) than in the freshwater-irrigated soils, indicating that the high number of resistant bacteria that enter the soils from the TWW are not able to compete or survive in the soil environment and that they do not significantly contribute ARG to soil bacteria. This strongly suggests that the impact of the TWW-associated bacteria on the soil microbiome is on the whole negligible, and that the high levels of AR bacteria and ARGs in both the freshwater- and the TWW-irrigated soils are indicative of native AR associated with the natural soil microbiome.