Human ingestion of antimicrobial residues in dairy products is a problem of public health. In this study we evaluated antibacterial effectiveness of aqueous and ethanolic leaf extracts of Mangifera indica L. as a natural alternative for reduction of Staphylococcus spp. biofilms. Eight Staphylococcus spp. strains from cows with mastitis and the Staphylococcus aureus strain (ATCC 25923) were evaluated. High performance liquid chromatography indicated the presence of phenolic compounds in the aqueous extract. Agilent Technologies gas chromatography revealed the presence of phenolic compounds, carbohydrates, and gallic acid in ethanolic extract. The tannin contents were 2.27 ± 0.01 and 2.67 ± 0.01% for ethanolic extract and aqueous extract, respectively. Extracts were evaluated in agar diffusion test and the size of inhibition zones were compared. The ethanolic extract was the most effective, showing minimum inhibitory concentrations and minimum bactericidal concentrations between 1.8–7.5 and 15.1–45.3 mg/ml, respectively. After tannin removal, the extracts were less effective, indicating these secondary metabolites as one of the main antibacterial compounds. The ethanolic extract at 45.3 mg/ml reduced mature biofilms in teat rubber (average: 3.91 log reduction) and stainless steel (average: 3.87 log reduction) after 5 min of contact. The extracts of M. indica leaves represent natural alternatives against Staphylococcus spp. strains and the ethanolic extract shows potential as a natural sanitizer.