Lactic acid bacteria produce metabolites with antagonistic activity against other bacteria. However, growth conditions and conservation methods may reduce the viability and antimicrobial activity of lactic acid bacteria. This study evaluated the effects of fermentation substrate, lyophilization (freeze-drying) and refrigeration on the viability and antimicrobial activity of Weissella confusa strain and its metabolites against pathogens responsible for bovine mastitis. W. confusa strain was grown in MRS broth and milk supplemented with yeast extract and glucose (MYEG). The collected fractions were preserved by lyophilization or under refrigeration at 4ºC. Every seven days, the viability of W. confusa strain and the stability of its metabolites were evaluated against Staphylococcus aureus and Streptococcus agalactiae by disc diffusion assays. In both fermentation substrates, the combination of lyophilized strain and metabolites retained antimicrobial activity against the two pathogens for 42 days. Also, W. confusa strain retained adequate viability and antimicrobial activity when grown in MYEG and stored under refrigeration conditions. It was concluded that MYEG and refrigeration are acceptable low cost options to preserve the viability of W. confusa for its potential commercial use in the prevention and treatment of bovine mastitis.