Background and Objective: Bovine mastitis, particularly caused by Staphylococcus spp., poses challenges in dairy production. This study assessed the antimicrobial activity of various antibiotics on strains isolated in Ecuador. Materials and Methods: Milk samples from cows with mastitis were collected in Ecuador, isolating and identifying Staphylococcus spp. The strains were tested against antibiotics using the disk diffusion method. Results: Streptomycin exhibited the highest effect, with an average halo diameter of 27.86 mm (30µg), followed by neomycin with 25.40 mm (30µg) and gentamicin with 24.80 mm (30µg). However, high resistance to betalactams was observed, with penicillin reaching only 23.26 mm (30µg). These findings suggest inefficacy in current therapeutic treatments, especially in mastitic cases treated with the analyzed drugs. It is crucial to consider these findings to adapt therapeutic strategies addressing bacterial resistance and improving treatment efficacy in dairy cattle. Conclusion: Continuous monitoring of antimicrobial sensitivity is essential for optimizing livestock health and productivity, adjusting treatment protocols according to bacterial resistance evolution.