Mushrooms are a rich source of bioactive compounds, including antimicrobial agents with significant potential against multidrug-resistant pathogens. These bioactive metabolites, such as polysaccharides, phenolic compounds, terpenoids, and sterols, exhibit diverse mechanisms of action, including disruption of microbial cell walls and interference with essential metabolic pathways. The extraction of these antimicrobial compounds requires a systematic approach involving soxhlet extraction, ultrasound-assisted extraction, microwave-assisted extraction, and supercritical fluid extraction. Standard microbiological assays, such as agar well diffusion, broth microdilution, and time-kill kinetics, are employed to assess their antimicrobial efficacy against bacterial and fungal strains. Advanced techniques including minimum inhibitory concentration (MIC) determination and TLC studies also give more insights into their respective efficacy as well as application against few infectious microbes. This chapter highlights the extraction processes and the methods used to determine antimicrobial action, along with the bioactive compounds with antimicrobial activity from mushroom.