The present study was investigated to purify and characterize anti-tubercular and anticancer protein from Staphylococcus hominis strain MANF2 under mild stress condition of Mentha piperita L. Initially, the in vitro anti-tubercular activity of strain MANF2 was determined against Mycobacterium tuberculosis H37Rv using luciferase reporter phage (LRP) assay which showed relative light unit reduction (RLU) of >90 %. Further, MTT test revealed promising in vitro anticancer trait of strain MANF2 against lung (A549) and colon (HT-29) cancer cell lines. Mild stress of M. piperita L. was provided to strain MANF2 at lag and log phase of its growth and the protein production was optimized statistically using central composite design of response surface methodology. Results showed enhanced protein production in the medium containing yeast extract (0.5 % w/v) and glycerol (1.5 % v/v), being supplemented with M. piperita L. (1.5 % v/v at log phase of strain MANF2. Protein was purified using standard purification techniques and showed single homogeneous band on SDS-PAGE with nominal molecular mass of 51293 Da, as confirmed by MALDI-TOF MS/MS. The N- amino acid sequencing showed homology with proline dehydrogenase (ProDH), thus, the protein was proposed to be new ProDH-like protein in S. hominis. Further, LRP test revealed concentration dependent (10-50 μg/mL) in vitro anti-tubercular properties of purified protein with significant RLU reductions of 36.8 ± 0.3-78.5 ± 0.4 %. The IC values of purified protein against A549 and HT-29 cancer cells were calculated as 42.2 and 48.4 μg/mL, respectively. In conclusion, protein purified from strain MANF2 under mild stress of M. piperita L can certainly be implied as efficacious anti-tubercular and anticancer agents in future.