Avian colibacillosis, caused by Escherichia coli, remains a significant threat to poultry health and production, particularly in the context of rising antimicrobial resistance. Efficient and scalable vaccination strategies are needed to reduce economic losses and reliance on antibiotics. This study investigated the safety and immunogenicity of a novel single-dose in ovo vaccine candidate based on three inactivated E. coli strains formulated with cationic maltodextrin nanoparticles. The vaccine was evaluated in broilers under commercial hatchery conditions. In ovo administration was well tolerated and did not adversely affect hatchability, survival, growth performance, or feed efficiency. Vaccinated birds mounted a measurable serum immunoglobulin Y (IgY) response against E. coli from 14 days post-hatch, which persisted until slaughter age. Furthermore, when co-administered with routinely used live-attenuated viral vaccines, no interference with the immunogenicity of these vaccines was observed. These results demonstrate that the inactivated nanovaccine is safe, immunogenic, and compatible with an industrial-scale in ovo vaccination. The findings support its potential as a practical prophylactic approach to prevent avian colibacillosis in broiler production.