Background and Objective: Nowadays, the world is facing significant challenges in modern healthcare services because many antimicrobial agents have lost their effectiveness in treating infectious diseases primarily due to the development of microbial resistance. Mushroom species release various bioactive compounds and yet largely untapped resource of useful natural compounds. This study aimed to assess the primary screening for antimicrobial activity of selected Malaysian mushroom’s against Escherichia coli and Staphylococcus aureus.Methodology: The mycelia biomass extracts of the 8 collected Basidiomycetes mushrooms. Antibacterial activity was measured using Disc diffusion and Well diffusion methods against standard reference strains E. coli MTCC 740 and S. aureus MTCC 501. Results: The potential inhibition of Agaricus cupreobrunneus, which gave a reading of 4.6 cm for disc diffusion and 3.4 cm for well diffusion of inhibition against E. coli MTCC 740 and against S. aureus MTCC 501 that, gave a reading of 4 cm for disc diffusion and 3.1 cm for well diffusion approaches. The second highest bioactivity mushroom against E. coli MTCC 740 the highest inhibition was shown by extract of brown Agaricus bisporus which gave 3.5 cm and give 3.2 cm S. aureus MTCC 501 by disc diffusion. For well diffusion the zone of inhibition by same mushroom for E. coli MTCC 740 which gave reading 3.0 cm and gave 2.7 cm against S. aureus MTCC 501. Conclusions: The species studied showed potential inhibition of A.cupreobrunneus more than other mushrooms by disc diffusion method especially against E. coli more than S. aureus. Further studies are required to ascertain their toxicity against mammalian cells and potential side effects.