Eucalyptus oil nanoemulsion (EUCsingle bondNE) was evaluated as an eco-friendly disinfectant to improve hatchability and control bacterial contamination in ostrich hatcheries. Key pathogens identified in the current study included Pseudomonas species (39.39 %), Escherichia coli (36.36 %) and Staphylococcus species (36.36 %), most of which showed resistance to common antibiotics. EUCsingle bondNE exhibited potent antimicrobial activity against multidrug-resistant strains, with an IC₅₀ of 1.68 mg/mL in human fibroblast (HFB4) cells, confirming its safety and efficacy. In preliminary experiments, EUCsingle bondNE reduced E. coli and Salmonella contamination, resulting in hatch rates of 80 % and 70 %, respectively. No Eucalyptol (1,8-cineole) or alpha-pinene residue was detected in embryo livers. A field experiment on 30 ostrich eggs demonstrated a significant reduction in total bacterial counts on eggshells (P < 0.0001), complete elimination of E. coli (0 % isolation), and reductions of 30 % and 60 % in Pseudomonas species and Staphylococcus aureus isolations, respectively, outperforming a commercial disinfectant (TH4+). Results demonstrate the EUCsingle bondNE’s effectiveness and potential as a safer, natural alternative for controlling resistant bacterial contamination in ostrich hatcheries. This study advocates the use of cutting-edge nanotechnology in innovative, environmentally friendly disinfectants, minimizing chemical residues that threaten human health and mitigating the risks of the spread of antibiotic-resistant bacteria in the environment.