The increase of vancomycin-resistance due to co-colonization or co-infection with Enterococci (VRE) and Staphylococci (MRSA) in Algerian hospital settings is poorly reported, in which, few data of the molecular mechanism have been accessible on study of this resistance across the country. In this study, we investigate the epidemiology and antibiotic resistance of multi-drug resistant enterococcal and staphylococcal strains isolated from various clinical samples taken from hospitalised patients in Biskra hospital and outpatients within the study period from 2019 to 2021. All strains collected were identified using VITEK® 2 system, MRSA and VRE phenotypic confirmation by vancomycin and teicoplanin E-test were carried out. A total of 24,642 patients were analysed, of which 270 patients harboured staphylococcus including 80 MRSA and 135 Enterococci including 37 VRE. We identified 37 case patients for whom a single clinical specimen yielded both VRE and MRSA on culture. We carried out univariate comparisons between the 2 groups, then a multivariate logistic regression analysis. Results revealed that Staphylococcus aureus,E. faecium and E. faecalis strains are the main pathogens identified in this study. The majority of strains were detected in intensive care unit (ICU) (56.8%) followed by surgical wards (SW) 29.70%. The highest prevalence was found on pus (67,5%) followed by the blood culture (32.6%)The rate 52.2% of patients harboring both (MRSA) and (VRE) was male. Antibiotic susceptibility testing revealed that the resistance rate was high for the majority of antibiotic classes, including glycopeptides in which, MIC VAN > 256 μ g/mL and MIC TEC > 256 μ g/mL was observed for all resistant strains.