Enterococci are part of the normal intestinal flora of humans and animals but have also emerged as important pathogens responsible for serious infections in hospital and community acquired infections. The aim of this study was to investigate the prevalence of Enterococcus with their antimicrobial susceptibility pattern from patients of Mymensingh Medical College Hospital, Bangladesh during the period from July 2015 to June 2016. Samples were collected and identification of enterococcus was done by standard microbiological procedure and biochemical tests. Multiplex PCR was performed by using species specific primers for Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium), vanA, vanB, vanC1, vanC2/C3 genes for vancomycin resistance. A total of 105 enterococci were isolated from 1201 different clinical specimens (from urine (931), pus (175) and wound swab (95) respectively) from July, 2015 to June, 2016. The prevalence of enterococci was 12.34% in urine, 17.17% in pus and 1.58% in wound samples. Out of 105 enterococci, 80 (77.14%) were Enterococcus faecalis, 1 (0.95%) was E. gallinaram and 24 (22.86%) were other species. All the species were highly resistant to aminoglycosides (Gentamicin) (75.24%) and ciprofloxacin (73.33%). Regarding antibiotic sensitivity, all the enterococci were (100%) sensitive to linezolid and vancomycin followed by nitrofurantoin 93 (88.57%) and imipenem 89 (84.76%). No vancomycin resistant enterococci were identified by disk-diffusion method. But by PCR, vanB was found in 1/80 (1.25%) of the E. faecalis isolates and 01 (100%) of the E. gallinarum. vanC1 was detected in 1 (100%) of E. gallinarum isolates. The vanA and vanC2/C3 gene was not found in any isolates. For the first time, one isolate of E. gallinarum has been found harboring the vanB gene in our hospital. The presence of multidrug resistant enterococci should be considered as danger alarm for serious enterococcal infections and further study in large scale is needed.