Introduction. The emergence of multidrug-resistant Salmonella Typhimurium strains has increased the need for safe, alternative therapies from natural sources with antibacterial properties. Hypothesis/Gap Statement. There are no published data regarding the use of chitosan propolis nanocomposite (CPNP) either alone or in combination with antibiotics as antimicrobials against S. Typhimurium, especially in Egypt. Aim. This study evaluated the antibacterial activities of five antimicrobials [apramycin, propolis, chitosan nanoparticles (CNPs), chitosan propolis nanocomposite (CPNP) and CPNP +apramycin] against ten virulent and multidrug-resistant (MDR) S. Typhimurium field strains recovered from diarrheic rabbits through in vitro and in vivo study. Methodology. The expression levels of three virulence genes of S. Typhimurium strains were determined by quantitative reverse-transcription PCR (RT-qPCR) after exposure to sub-inhibitory concentrations of apramycin, propolis, CNPs, CPNP alone, and CPNP +apramycin. Additionally, 90 New Zealand rabbits were divided into control and experimentally S. Typhimurium-infected groups. The infected rabbits were orally administered saline solution (infected–untreated); 10 mg apramycin/kg (infected–apramycin-treated); 50 mg propolis/kg (infected–propolis-treated); 15 mg CPNP/kg (infected–CPNP-treated) and 15 mg CPNP +10 mg apramycin/kg (infected–CPNP +apramycin-treated) for 5 days. Results. The RT-qPCR analysis revealed different degrees of downregulation of all screened genes. Furthermore, the treatment of infected rabbits with CPNP or CPNP +apramycin significantly improved performance parameters, and total bacterial and Salmonella species counts, while also modulating both oxidative stress and altered liver and kidney parameters. Conclusion. This work demonstrates the use of CPNP alone or in combination with apramycin in the treatment of S. Typhimurium in rabbits.