Stenotrophomonas maltophilia (S. maltophilia) is a Gram-negative bacillus, an opportunistic pathogen, particularly among nosocomial infections. Multi-drug resistant strains are associated with very high rate of morbidity and mortality in severely immunocompromised patients. Present study was designed to evaluate the effect of biofield treatment against multidrug resistant S. maltophilia. Clinical sample of S. maltophilia was collected and divided into two groups i.e. control and biofield treated which were analyzed after 10 days with respect to control. The following parameters viz. susceptibility pattern, minimum inhibitory concentration (MIC), biochemical studies and biotype number of both control and treated samples were measured by MicroScan Walk-Away® system. The results showed an overall change of 37.5% in susceptibility pattern and 39.4% in biochemical study while 33.3% changes in MIC values of tested antimicrobials after biofield treatment. Further, the treated group of S. maltophilia has also shown a significant change in biochemical reactions followed by its biotype number as compared to control group. Biochemical reactions of treated group showed negative reaction to acetamide and positive reactions to colistin, glucose, adonitol, melibiose, arabinose, nitrate, oxidation-fermentation, raffinose, rhaminose, sorbitol, sucrose, and Voges-Proskauer as compared with control. The biofield treatment showed an alteration in MIC values of amikacin, amoxicillin/K-clavulanate, chloramphenicol, gatifloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotetan, ticarcillin/K-clavulanate, trimethoprim/sulfamethoxazole. Altogether, data suggest that biofield treatment has significant effect to alter the sensitivity pattern of antimicrobials and biotype number against multidrug resistant strain of S. maltophilia.