BACKGROUND: Gram-positive Streptococci is a huge group of different species that are classified based on its hemolytic effect besides the C-substance in the cell wall. This study focuses on the investigation of the prevalence and genetic basis of resistance to macrolides, lincosamides, and streptogramins (MLS) in α- and β-hemolytic Streptococci. METHODS: Streptococcal isolates were identified and their resistance was assessed to MLS antibiotics through phenotypic analysis and genotypic screening of resistance genes. Isolates were also tested for susceptibility to antiseptics/disinfectants. The correlation between high MLS antibiotic resistance and reduced susceptibility to biocides was assessed. Efflux pump activity in the most resistant isolates (to both MLS antibiotics and biocides) was investigated. RESULTS: The susceptibility testing indicates an increasing resistance to MLS, particularly macrolides (erythromycin, azithromycin, and clarithromycin) and lincomycin. By screening the resistance, the most predominant phenotype is the constitutive (cMLS) one, while the erm genes, particularly ermB, are the most detected genotype. Furthermore, the esterase-encoding gene ereA is widely distributed in the streptococcal isolates. By evaluating the minimum inhibitory concentrations (MICs) to different biocides, there was a strong relation between the increased MIC values to both MLS antibiotics and tested biocides. This can be attributed mainly to the transferable ermB gene and the enhanced bacterial efflux. CONCLUSIONS: A significant correlation exists between reduced biocide susceptibility and resistance to MLS antibiotics. Elevated efflux pump activity in MLS-resistant isolates suggests efflux mechanisms may contribute to dual resistance to antibiotics and biocides. However, cross-resistance is primarily driven by the horizontally transferable ermB gene, which confers resistance by targeting the 50S ribosomal subunit.