The antimicrobial activities, free radical scavenging capacity, the content of total phenols, and total flavonoids of primary and secondary distilled bamboo vinegar were investigated. In addition, the primary and secondary distillation bamboo vinegar was analyzed for the main bioactive compounds by HPLC. The results indicated that the bamboo vinegar displayed strong antimicrobial activities against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Canidia albicans and Aspergillus niger, and free radical-scavenging properties. However, antioxidant capacity and antimicrobial activities of secondary distilled bamboo vinegar were significantly lower than these of primary distilled bamboo vinegar. The HPLC fingerprint showed that the peak numbers and areas of secondary distilled bamboo vinegar at 254nm and 270nm were obviously lower than these of primary distilled bamboo vinegar.