The use of antibiotics is the preferred therapy for bacterial diseases. However, overusing antibiotics has led to the development of antibiotic resistance in bacteria, which is now a major public health concern. Therefore, in this study, the performance of lysozyme (LYZ)/tracheal antimicrobial peptide (TAP)-based tissue-specific expression antimicrobial plasmids (TSEAP) have been evaluated in the treatment of mastitis in mice. The results show that LYZ/ and TAP-based TSEAP could effectively reduce the clinical symptoms caused by Staphylococcus sciuri, Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa-induced mastitis. In addition, the studies of behavioral tests, parameters of weight growth, blood biochemistry, and organ coefficients comprehensively indicate that the transfection of LYZ/TAP-based TSEAP is safe in mice. Taken together, LYZ/TAP-based TSEAP have broad-spectrum antibacterial activity and may provide new insight for the non-antibiotic treatment of bacterial diseases.