Objective: Breast cancer is the most common cancer faced by women around the worldwide. The Estrogen receptor alpha (ERα) has been playing a major role in the stimulation of breast cancer. The present study aims to identify the anticancer activity of crude extract of Bacillus subtilis against breast cancer cell line by in vitro and in silico methods. Methods: The soil samples were collected from different regions in the reserve forests of Western Ghats of Nilgiris district in Tamil Nadu, India. Isolation of bacterial strain from the collected soil samples was performed by serial dilution method. Identification of bacterial strain was done by 16S rRNA sequencing analysis. The anti-cancer potential of crude extract of bacterial strain was tested against breast cancer cell line (MCF-7) by MTT assay. Further, the bioactive compounds found in the crude extract of bacterial strain was identified by GC-MS and the identified compounds was subjected to in silico docking studies against the targeted breast cancer protein Estrogen receptor alpha (ERα). Result: The bacterial strain isolated from the soil sample by serial dilution method was identified as Bacillus subtilis by 16S rRNA analysis. In vitro anti-breast cancer analysis of crude extract of Bacillus subtilis showed potential activity against MCF-7 breast cancer cell line with the IC50 value of 100μg/ml. The GC-MS analysis of the ethyl acetate extract of Bacillus subtilis showed twenty-six bioactive compounds. The compound Metaraminol showed maximum docking score -7.27 Kcal/mol against the target protein. Conclusion: The crude extract of soil bacterium showed potent anti-cancer activity against breast cancer cell lines. Further, the bioactive compounds showed good binding interactions with the virulence factor of breast cancer. Thus, the compounds of soil bacterium Bacillus subtilis could effectively used as leads for developing drugs against breast cancer.