Introduction: The aim of present study is to investigate the antimicrobial activity of Withania somnifera (RUBL-20668) and Cenchrus setigerus (CAZRI-76) extracts in order to use it as a possible source for new antimicrobial substances against important human pathogens. Method: The crude extracts of different parts (root, stem, leaf and seed) of C. setigerus and (Unripen fruit, ripen fruit, calyx) of W. somnifera and were successively extracted with polar to non polar solvents using soxhlet assembly. The extracts were then screened for their antimicrobial activity in-vitro against one gram positive bacteria (Bacillus subtilis), two gram negative bacteria (Pseudomonas aeruginosa and Enterobactor aerogens) and one fungus (Aspergillus flavus) by disc diffusion assay. Serial dilution method was used to determine minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC). Result: Water extract of stems of C. setigerus (IZ-21.83 ± 0.24 mm, AI- 0.780) and chloroform extract of calyx of W. somnifera (IZ-16.17 ± 0.24 mm, AI- 1.078) showed highest activity against B. subtilis. Conclusion: The demonstration of broad spectrum of W. somnifera and C. setigerus may help to discover new chemical classes of antibiotic substances that could serve as selective agents for infectious disease chemotherapy and control.