This study was conducted to investigate the potential use of Cordyceps militaris spent mushroom substrate as a natural antimicrobial agent. The fruiting body and spent mushroom substrate of C. militaris were extracted into 80% ethanol, and the antimicrobial activity was compared. Both extracts (1 mg/disc) exhibited clear antimicrobial activity against Pseudomonas aeruginosa in the agar diffusion assay. The MIC values of the fruiting body and spent substrate extracts against P. aeruginosa were determined to be 0.4 mg/ml and 0.6 mg/ml, respectively, indicating a bacteriostatic action. The inhibitory effects of the C. militarisspent mushroom substrate extract on biofilm formation of P. aeruginosa were determined by biofilm biomass staining and qRT-PCR analysis. The biofilm biomass and cell growth of P. aeruginosa in the cultures treated with 0.2–2.0 mg/ml of extracts were significantly decreasedin a concentration-dependent manner. The qRT-PCR analysis showed that the lasI and lasR gene expression associated to quorum sensing (QS) in the cultures treated with 0.2–2.0 mg/ml of extracts were suppressed in a concentration-dependent manner. Based on the above results, it can be concluded that extracts from C. militaris spent mushroom substrate can be used as an antimicrobial agent derived from natural materials, as demonstrated by the bacteriostatic action and inhibition of biofilm formation of P. aeruginosa.