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BACKGROUND Stenotrophomonas maltophilia is a gram-negative bacillus, multidrug resistant (MDR) opportunistic pathogen, which is normally present in hospital surroundings. It has been one of the leading causes of nosocomial infections due to risk factors such as extended intensive care unit (ICU) stays and multiple invasive procedures. In this study we wanted to assess the antibiotic sensitivity pattern with various antimicrobial agents i.e. levofloxacin, minocycline, ceftazidime, chloramphenicol, & ticarcillin-clavulanic acid with special focus on trimethoprim-sulfamethoxazole (TMP-SMX). METHODS In vitro analysis was conducted on 164 Stenotrophomonas maltophilia strains isolated from blood and respiratory tract from January 2016 to November 2020. Antibiotic susceptibility and minimum inhibitory concentration (MIC) testing for trimethoprim-sulfamethoxazole (TMP-SMX), levofloxacin (LVX), ticarcillin - clavulanic acid (TIM), and minocycline (MIN) were performed using Vitek 2, as per clinical and laboratory standards institute (CLSI) guideline. RESULTS A total of 164 S. maltophilia were isolated. Out of the 164 S. maltophilia isolates, 26 (16 %) were isolated from blood, 114 (70 %) were isolated from respiratory samples, 20 (12 %) from pus & tissue, and 4 (2 %) from urine. Out of the 164 patients, 130 (80 %) were males and 32 (20 %) were females. Maximum patients were above 50 years of age 93 (56 %) followed by 20 - 50 years 55 (34 %). Out of the 164 patients, 67 (40 %) were admitted to wards, 92 (56 %) were in ICU and 5 (3 %) were seen in out-patient department (OPD). A total of 90 % strains were sensitive to trimethoprim-sulfamethoxazole (TMP-SMX). Total 91 % strains were sensitive to levofloxacin. CONCLUSIONS S. maltophilia is emerging as a significant nosocomial pathogen, with a growing rate of isolation. Trimethoprim- sulfamethoxazole (TMP-SMX) is still the drug of choice, but resistance to it has been reported. KEYWORDS Minimum Inhibitory Concentration, Trimethoprim-Sulfamethoxazole (TMP-SMX), Levofloxacin