<p>The study encompassed the isolation of <i>Staphylococcus aureus</i> (<i>S. aureus</i>) from bovine mastitic milk samples and its characterization for antibiotic sensitivity and biofilm production. The milk samples were initially screened for <i>Staphylococcus</i> spp. using cultural and biochemical tests, and were confirmed to species level using biochemical tests, Polymerase Chain Reaction (PCR) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Antibiotic susceptibility was tested using the disc diffusion method, and biofilm formation was evaluated using the Modified Congo Red Agar assay (MCRA assay) and Microtitre based Biofilm Assay (MtBA). Out of 112 samples, 98 showed bacterial growth, of which 18.36% were presumptively identified as <i>Staphylococcus</i> spp. through cultural and biochemical tests. These isolates were further confirmed as <i>S. aureus</i> using biochemical tests, PCR and MALDI-TOF. All the isolates were tested against 25 antibiotics and the results demonstrated predominant resistance to cefoxitin (83.3%), ampicillin and penicillin G (each 77.8%), while most were sensitive to enrofloxacin and vancomycin (each 83.3%). The two biofilm detection methods employed in the study showed significant agreement (<i>p</i> = 0.021) in identifying strong biofilm producers. Concurrently, a significant association (<i>p</i> = 0.0123) was found between methicillin resistance and biofilm formation, consistent with MRSA phenotypes exhibiting a greater predisposition for biofilm production. Overall, the study revealed the occurrence of multidrug-resistant <i>S. aureus</i> in bovine mastitis, with significant biofilm-forming ability particularly among the methicillin-resistant isolates. These findings address the imperative for including biofilm assessment alongside the conventional antimicrobial susceptibility test in managing persistent Staphylococcal mastitis.</p>