A sudden emergence of Stenotrophomonas maltophilia as a primary pathogen both in immunocompromised and immunocompetent individuals has raised a serious concern, as it is associated with significant case fatality ratio. We intended to study the clinico-microbiological profile of S. maltophilia isolates from various samples and outcome of the infections in a tertiary healthcare center, Pune, India. This is an observational cross-sectional study was conducted from January 2021 to June 2022 at Department of Microbiology of a tertiary care Centre in Pune, India. Of the 12049 samples received for culture, S. maltophilia was isolated in 57 samples. Only 42 samples with pure growth of S. maltophilia were included in the study with 15 excluded due to mixed growth. All isolates were confirmed by VITEK-MS (bioMerieux, SA, France) which uses Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) technology. Of the 42 isolates, majority were isolated from pus(28.6%) and most of patients (61.9%) were from acute health care settings. The isolates had high susceptibility to Cotrimoxazole (85.7%) and Minocycline (85.7%) and low susceptibility to Ceftazidime (45.2%). A case fatality rate of 7.1% (3/42 cases) was noted and 39 cases were discharged after complete treatment. All the three fatal cases were susceptible to levofloxacin, ciprofloxacin, cotrimoxazole and minocycline and all three fatal cases were resistant to ceftazidime. S. maltophilia has recently shown an increase in nosocomial infections especially in acute healthcare settings like ICU and other critical care wards. The isolates of the present study had high susceptibility to trimethoprim-sulfamethoxazole (TMP-SXT) and Minocycline and low susceptible to Ceftazidime.