Given the high cost and non‐renewability of mineral‐based fertilizers, there is increasing interest in the innovative use of manure‐based materials, such as poultry litter (PL). However, manure‐based fertilizers add both nutrients and microbes to the soil, including antibiotic‐resistant Escherichia coli (AREc). PL soil amendment impact on AREc in corn fields was evaluated in a randomized field experiment (May–October 2017). Two winter cropping systems (fallow and cover crop) were assigned to whole plots, with three spring‐applied fertilizer treatments (untreated control [UC], PL, and commercial fertilizer [CF]) assigned to subplots. Soil was collected from 0 to 15 cm on days 0, 7, 28, 70, 98, and 172 post‐treatment applications. Samples were cultured for the enumeration and prevalence of generic, tetracycline‐resistant (TETr), third‐generation cephalosporin‐resistant (3GCr) E. coli isolates, and extended spectrum beta‐lactamase (ESBL)‐producing Enterobacteriaceae. PL soil amendment significantly (p < 0.05) increased the levels of generic E. coli, TETr E. coli, and 3GCr E. coli on days 7 and 28 compared to UC or CF. Beyond day 28, AREc did not significantly (p > 0.05) differ by fertilizer treatment and returned to baseline on day 70. ESBL‐producing Enterobacteriaceae were detected from 16 samples, mostly on day 70. Cover crop significantly decreased TETr E. coli concentration on day 28, with no significant effects on the prevalence of 3GCr E. coli and ESBL‐producing Enterobacteriaceae compared to no cover crop. All ESBL‐producing Enterobacteriaceae and 79% of the 3GCr E. coli isolates were positive for blaCTX‐M gene by polymerase chain reaction. Results show that PL soil amendment transiently increases the levels of AREc compared to mineral fertilizer.