Field application of beef cattle feedlot runoff may transport manure‐borne microbes and antibiotic resistant bacteria to agricultural soils eventually impacting deeper soils and groundwater. To evaluate this potential, total soil, antibiotic resistance (AR), and fecal indicator bacteria (Escherichia coli and Enterococcus) and the presence/abundance of AR genes were examined to a depth of 1.8 m in an agricultural field receiving long‐term application of feedlot runoff and compared to a nearby pasture receiving no runoff. While plate counts of total soil bacteria and cefotaxime‐resistant, erythromycin‐resistant, and tetracycline‐resistant bacteria decreased with depth on both fields (p < 0.001) by an average 2‐log10 colony forming unit g−1 to 1.8‐m depth, field differences were only observed with greater abundances of total soil and erythromycin‐resistant bacteria (p ≤ 0.026) in the runoff‐amended versus control field soils. Soil bacterial and fecal indicator bacterial isolates evaluated phenotypically for resistance to 12 antibiotics varied in range and sensitivity. Using a culture‐independent approach, erm(C) and tet(Q) were detected using polymerase chain reaction in 31% and 58% of runoff‐field samples throughout the soil profile. Detection of erm(C) and tet(Q) in the control field soil profile was less frequent (0% and 11%, respectively). Two other genes, erm(A) and tet(X) were not detected in any soil samples. Based upon these results, long‐term applications of beef cattle feedlot runoff may increase the total abundance of microorganisms in the surface and shallow soil, but the relative enrichment of AR was dependent upon the type of resistance evaluated and, more specifically, the genes targeted for analysis.