Background: Macrolide resistance has increased worldwide among Gram-positive cocci including staphylococci, particularly after the irrational use of macrolides during the COVID-19 pandemic. Scarce data exists about the situation in Zagazig University Hospitals. Aim: To detect different macrolide resistance phenotypes and genotypes among staphylococcal clinical isolates in Zagazig University Hospitals, one of the tertiary hospitals in Egypt. Methods: Antibiotic susceptibility of ninety-two staphylococcal isolates collected from various clinical specimens, was carried out against erythromycin, azithromycin, and clindamycin by disc diffusion method. The D-test was applied to detect inducible macrolide, lincosamides, and streptogramin type B resistance phenotype (iMLSB). Molecular detection of major genes coding for macrolide resistance, including erythromycin ribosomal methylase (ermA, ermB, and ermC), and macrolide-streptogramin resistance gene (msrA) was performed using PCR. Results: Out of 92 staphylococcal isolates, 37 isolates (40.2%) showed macrolide resistance. The iMLSB phenotype was identified in 32.4% of the resistant isolates with a rate of 43.7% among methicillin-resistant Staphylococcus aureus (MRSA), meanwhile, constitutive resistance was detected in 43.2%. The investigated resistance genes were detected in a total of 89.2% of resistant isolates where the ermC was the most frequent (54.1%), followed by the msrA gene (45.9%), the ermA gene (16.2%), and the ermB (5.4%). However, none of the examined genes showed a statistically significant relationship with the resistance phenotypes (P > 0.05). Conclusion: Our finding revealed increased macrolide resistance, particularly the inducible phenotype among MRSA isolates with wide dissemination of macrolide resistance genes, necessitating continuous monitoring.