Background: An important public health issue is the rising frequency of infections brought on by macrolides, lincosamides, and streptogramin-B (MLS-B)-resistant staphylococci. Clindamycin can cause resistance and therapeutic failure when used to treat infections caused by inducible (iMLS-B) strains. Objectives: The goal is to identify staphylococcal isolates resistant to MLS-B based on phenotype and genetics. Subjects and Methods: This study was scheduled to run from September 2021 through August 2022. Clinical samples were collected from patients admitted to Mansoura University Hospitals with varying signs and symptoms of infection and cultured on the appropriate culture media. Colony morphology, Gram stain, biochemical response, and VITEK2 were used to identify isolated colonies. The VITEK2 system was used to test for antibiotic susceptibility. Results: Bacterial growth was detected in 753 of the 2450 samples analyzed. 350 of them were Gram-positive bacteria. Of the 350 samples, 200 were staphylococci, and 50 of them were staphylococci with MLS-B resistance. According to a D-test, 53.2% of MLS-B phenotype had constitutive (cMLS-B), 28.2% had iMLS-B, and 18.6% had MS. Staphylococcus aureus exhibited a 9.3% MS phenotype, 15.7% iMLS-B, and 18.7% cMLS-B. ErmA, ermB, ermC, and msrA genes yielded 10%, 32%, 74%, and 4.0%, respectively. In coagulase-negative staphylococci (CoNS), cMLSB was the most common trait and ermC gene dominated, followed by the ermB gene. In S. aureus, the ermC gene dominated the iMLSb phenotype (55%). Conclusion: The dominant genes among MLS-B resistant isolates are ermC and ermB, while cMLS-B is the prevalent phenotype. To prevent therapeutic failure, it is crucial to identify the iMLS-B phenotype by D-test and find the resistance genes prior to clindamycin administration.