Breast cancer is a serious global health problem, and investigation of innovative therapeutic approaches in its treatment is important to increase survival. Combination therapy targets more than one mechanism simultaneously and has recently emerged as an effective treatment strategy by using different therapeutic agents together. The purpose of this study was to determine the combined effects of the conventional chemotherapeutic agent 5-Fluorouracil (5-FU) and the HSP90 inhibitor XL-888 on breast cancer cell lines. MDA-MB-231 and MCF-7 cells were subjected to varying concentrations of XL-888 and 5-FU as individual treatments and in combination. The MTT test was employed to determine cell viability, and the Chou-Talalay technique was utilized to compute combination indices. Contrary to expectations, the HSP90 inhibitor XL-888 and 5-FU coadministration showed antagonistic effects in MDA-MB-231 and MCF-7 breast cancer cells. The results highlight the importance of careful consideration when combining these agents in breast cancer treatment regimens because their co-administration may not produce the expected synergistic results. The implications of the present research are anticipated to contribute to the developing of enhanced and focused treatment modalities for various cancers, with a particular emphasis on breast cancer.