Aims: To study virulence factors and antifungal susceptibility profile of C. glabrata isolated from various clinical specimens. Study Design: A total of 175 C. glabrata spp. isolated from various clinical specimens were included in the study. Place and Duration of Study: Department of Microbiology, Rural Medical College, Pravara Institute of Medical Sciences (PIMS), Loni, Maharashtra, India, between March 2008 to March 2013. Methodology: C. glabrata was identified by sugar assimilation and fermentation tests and colony color on Hichrome Candida agar. HiCandida identification kit supplemented the identification of the isolates. The virulence markers studied were production of extracellular hydrolytic enzymes (phospholipase, proteinase and coagulase), haemolytic activity and biofilm formation. The antifungal susceptibility profile of C. glabrata isolates was determined by Hicomb minimum inhibitory concentration (MIC) test. The antifungal agents used were amphotericin B (range 0.002-32 μg), fluconazole (range 0.016-256 μg), itraconazole (range 0.002-32 μg) and ketoconazole (range 0.002-32 μg). Results: Maximum number of isolates were obtained from blood culture (36%) followed by urine sample (29.7%). ICU stay followed by HIV infection were the main predisposing factors found to be associated with C. glabrata infection. A total of 53 (30.2%) C. glabrata Research Article British Microbiology Research Journal, 4(1): 39-49, 2014 40 isolates showed phospholipase activity. Proteinase production was seen in 56 (32%) isolates. 48 (27.4%) isolates were coagulase positive. Haemolytic activity was noted in 43 (24.5%) isolates. Most of C. glabrata produced βtype of haemolysis on sheep blood SDA agar. Biofilm forming ability was noted in 68 (38.8%) isolates. Maximum isolates were resistant to fluconazole (46.8%) and ketoconazole (46.8%) followed by itraconazole (45.7%). Amphotericin B resistance was seen in 58 (33.1%) isolates. Conclusion: Once considered as a non pathogenic human commensal, C. glabrata has emerged as an important pathogen in various clinical types of candidiasis. C. glabrata is innately resistant to antifungal drugs and various antifungal mechanisms of the body. Present research data available is not satisfactory to understand the pathogenic and other mechanisms involved in the transition of C. glabrata from nonpathogenic commensal to a potential pathogen. Therefore more research studies are needed to explain pathogenesis, host-pathogen interaction and other survival properties of this emerging pathogen.