The antibacterial activity of acetone, methanol, and petroleum ether extract of Daedaleopsisflavida was investigated against four human pathogenic bacteria: Escherichia coli (ATCC:8739), Pseudomonas aeruginosa (ATCC:9027), Bacillus subtilis (ATCC:6051) and Salmonella enterica (ATCC:14028). Minimal inhibitory concentration (MIC) was evaluated for each extract. Antibacterial activity was performed using agar well diffusion assay. This is the first report investigating the antibacterial activity of this mushroom. Petroleum ether and methanol extract significantly inhibited E. coli and P. aeruginosa as compared to the control (Streptomycin) while inhibition of S. enterica and B. subtilis was more or less the same for petroleum ether while methanol extract exhibited lesser activity. Acetone extract, however, exhibited more significant activity as compared to control, petroleum ether and methanol extract. Significant inhibitory activity was observed against P. aeruginosa (23.67± 0.58 mm) followed by E. coli (19.33 ± 1.15 mm), B. subtilis (18.00 ± 1.00 mm) and comparatively lower activity against S. enterica (15.33 ± 0.58 mm). Further investigation of acetone extract would reveal the bioactive compound.