Background Avian pathogenic Escherichia coli (APEC) are considered a growing health problem to both poultry and the public, particularly due to its multi-drug resistance. Zinc oxide nanoparticles (ZnO-NPs) are a promising multi-benefit candidate. This study focused on boosting the antimicrobial effect of the chemically synthesized ZnO-NPs using Polyethylene glycol-6000 (PEG-6000) and evaluating their potential to recover the sensitivity of Florfenicol and Streptomycin-resistant APEC to these drugs in a concentration range of 0.1–0.4 mg/mL. Four samples of ZnO-NPs were formulated and tested microbiologically. Results The physicochemical characterization showed well-crystallized spherical in situ synthesized ZnO-NPs using PEG-6000 (surfactant) and ethanol (co-surfactant) of ∼19–67 nm particle size after coating with PEG-6000 molecules. These ZnO-NPs demonstrated a strong concentration-dependent antibacterial effect against multidrug-resistant APEC strains, with a minimum inhibitory concentration of 0.1 mg/mL, Combining PEG-6000 coated in situ synthesized ZnO-NPs and Florfenicol induced 60% high sensitivity (30 mm inhibitory-zone), 30% intermediate sensitivity, and 10% resistance against APEC strains. The combination with Streptomycin revealed 50% high sensitivity, 30% intermediate sensitivity, and 20% resistance with a 20 mm maximum zone of inhibition using agar well diffusion test. Conclusion In situ preparation of ZnO-NPs using PEG-6000 and ethanol followed by coating with PEG-6000 enhanced its antibacterial activity in minimum inhibitory concentration and regained the efficacy of Florfenicol and Streptomycin against APEC, referring to a non-antibiotic antimicrobial alternative and an effective combination regimen against multidrug-resistant APEC E. coli in veterinary medicine.