Mushrooms are valued by people worldwide, gradually for their nutritive value and medicinal properties. Many medicinal effects of mushrooms have been reported recently, including anti-tumor, antibacterial, anti-diabetes, anti-oxidation, and other functions. Especially because of their antibacterial activity, medicinal mushrooms have been tested in clinical trials for the therapy of bacterial infection. Polysaccharides are one of the main bioactive components in medicinal mushrooms, which have potential medicinal value. Here, we introduce ultrasonic-assisted to separate and concentrate medicinal mushroom polysaccharides. Besides, antibacterial activities are measured by disk diffusion and macrodilution assay. Experimental methods are described particularly in this chapter.