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The emergence of antimicrobial resistance to commonly used antibiotics has necessitated the development of new antimicrobial products. Crude extracts produced by actinomycetes contain antimicrobial metabolites that can inhibit bacterial growth. The objective of our study was to evaluate the antimicrobial activity of crude extracts (Caat1-54 and CaatP5-8) produced by actinomycetes against isolates of Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, and Streptococcus uberis, which were obtained from the milk of cows affected by mastitis in 23 dairy herds. Twenty isolates of each bacterial species were used to define minimum inhibitory concentrations (MIC) of both crude extracts and ceftiofur (positive control). The MIC 50 and MIC 90 were defined at the concentration required to inhibit the growth of 50 and 90% of bacterial isolates tested, respectively. The MIC results were evaluated by survival analysis. Staphylococcus aureus isolates presented MIC 90 of Caat 1-54 ≥6.25 µg/mL, ceftiofur ≥12.5 µg/mL, and Caat P5-8 ≥100 µg/mL. Streptococcus uberis presented MIC 90 of ceftiofur ≥0.39 µg/mL, Caat 1-54 ≥50 µg/mL, and Caat P5-8 ≥100 µg/mL. Staphylococcus chromogenes isolated from subclinical mastitis presented MIC 90 of Caat 1-54 ≥0.78 µg/mL and ceftiofur and Caat P5-8 of ≥6.25 and ≥100 µg/mL, respectively. Streptococcus dysgalactiae isolated from clinical mastitis presented similar MIC 90 values between antimicrobials tested (ceftiofur, Caat 1-54, and Caat P-58), but these values (≥100 µg/mL) were higher than the values obtained from other pathogens evaluated in the present study. Our results indicate that Caat 1-54 and Caat P5-8 crude extracts present in vitro antimicrobial activity against isolates of Staph. aureus, Staph. chromogenes, Strep. dysgalactiae, and Strep. uberis isolated from clinical and subclinical mastitis., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)