Breast cancer resembles the second-highest incidence rate of cancers worldwide with highly remarkable incidence and mortality rates. Recently, the main target behind the current study is to reduce the toxicity along with targeted delivery of different drugs separately and simultaneously to enhance the treatment options for breast cancer. A promising strategy for overcoming poor drug delivery is to recruit the evacuated bacterial cells which are known as Bacterial Ghosts (BGs) for drug delivery. The present study focused on formulating a new complex delivery system using a highly purified and safety-confirmed BGs of Salmonella Typhimurium ATCC 1403 strain loaded with class II HDACI (Belinostat) associated with nano-emulsion droplets of constant and non-toxic natural compound (Betaine), a glycine with three additional methyl groups. The formulated new complex delivery system of BGs was tested on two breast cancer cell lines, ER-positive ZR-75-1 and TNBC, MDA-MB-231 respectively. Using WST-1 proliferation assay, our data reports a combination index (CI) <1 indicating synergistic effect between Betaine nano-emulsion at low concentrations with Poly dispersity index less than 0.5 and BGs loaded with Belinostat per constant count of BGs CFU/ml. In comparison with treatment of either Betaine or Belinostat alone, the viability inhibition and apoptosis induction of both tested MDA-MB-231 and ZR-75-1 cells were amplified in the combined treatment of 72 hours. Furthermore, the achieved delivery showed a significant downregulated expression of angiogenesis-related markers such as VEGF and TSP-1 at concentrations of 1-mM Betaine nano-emulsion and 0.2-µM Belinostat after 24 hours of treatment. In conclusion, the proposed study introduced a new formulated delivery system of BGs with synergistic mechanistic effect of two different anti-cancer compounds as a promising strategy to inhibit breast cancer cell lines proliferation and angiogenesis. Citation Format: Sara H. Deghedy, Hoda E. Mahmoud, Ahmed A. Hussein, Mohammed A. El-Kholy, Passent M. Gaafar, Mai M. Ali, Ahmed S. Sultan. Synergistic effect of bacterial ghosts loaded with belinostat and betaine nano-emulsion: A potential treatment strategy for breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 834.