The bacterium Staphylococcus can cause various health problems, particularly in hospitalized patients. Therefore, the current study aimed to isolate methicillin-resistant Staphylococcus aureus (MRSA) strains, test their capability to form a biofilm, and detect genes related to virulence and biofilm formation. Bacterial isolates were collected from the King Faisal Specialist Hospital and Children’s hospital in Taif Governorate, Saudi Arabia, and identified using primers for mecA and nuc1. They were tested for resistance against twelve widely distributed antibiotics and biofilm formation capability. The MRSA isolates were tested for fnbA, fnbB, and SCCmec. Among 100 isolates, 24 were identified as Staphylococcus aureus, and most of them were MRSA. Most isolates were resistant to cefrizine and cefepime (96%). The isolates showed higher resistance to amoxicillin and ampicillin (92%), followed by aztreonam (83%). Two isolates, S15 and S17, were high-grade positive for biofilm formation, 62.5% were medium-grade, and 20.8% were low-grade positive. Two of the isolates, S11 and S16, tested negative for biofilm formation. Furthermore, mecAI. ncu1 was found in all of the isolates, except S11. Most isolates had SCCmecIII and SCCmecV. All isolates were habituated to fnbB, while fnbA was not found in S3 and S11. These results indicated that PCR techniques offer rapid, simple, and accurate determination of the genetic profile and biofilm production capability of MRSA, and can be used in clinical diagnosis as well as to monitor the spread of antibiotic-resistant S. aureus strains.