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This study was conducted to determine the in vitro antibacterial effect of an ethanolic Morus alba L. (mulberry) leaf extract against Escherichia coli and Staphylococcus aureus isolated from mastitis dairy cows and against E. coli ATCC 25922 and S. aureus ATCC 25923 reference strains. The antibacterial efficacy was evaluated by a microdilution method to determine the MIC, MBC, and a time-kill assay by using azithromycin as a positive control. The data were analyzed nonparametric tests with a significance level of p<0.05. The quercetin content of the mulberry leaf extract was 83.187 ± 0.272 µg/g dried plant material, as determined by HPLC. The extract had MICs of 10, 20, 10, and 10 mg/mL and MBCs of 20, 20, 20, and 10 mg/mL for E. coli ATCC 25922, E. coli ECCM62, S. aureus ATCC 25923, and S. aureus SACM62, respectively. Quercetin had relevant MIC of 10 mg/ml and MBC of 20 mg/mL to the extract for both ATCC strains. Time-kill tests showed a complete elimination of S. aureus ATCC 25923 and S. aureus SACM62 after a 30-min exposure to 10 and 20 mg/mL M. alba extract. E. coli ATCC 25922 and E. coli ECCM62 showed reductions of 6.8 log CFU/mL after a 360 min exposure to 40 mg/mL M. alba extract. In this study, the inhibitory effect was stronger against S. aureus than against E. coli (p<0.05). Overall, the M. alba L. extract showed appreciable in vitro antibacterial efficacy against mastitis-causing E. coli and S. aureus strains and against reference ATCC strains
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