Study background: Staphylococci are widespread in nature, mainly found on the skin and mucous membranes. Staphylococcus aureus (S. aureus) is the key organism for food poisoning due to massive production of heat stable exotoxins. The current study was attempted to investigate the effect of biofield treatment on antimicrobial susceptibility pattern and biochemical characteristics of S. aureus (ATCC 25923). Methods: S. aureus cells were procured from MicroBioLogics in sealed packs bearing the American Type Culture Collection (ATCC 25923) number and stored according to the recommended storage protocols until needed for experiments. Revived and lyophilized state of ATCC strains of S. aureus were selected for the study. Both revived (Group; Gr. II) and lyophilized (Gr. III) strain of S. aureus were subjected to Mr. Trivedi’s biofield treatment. Revived treated cells were assessed on day 5 and day 10 while lyophilized treated cells on day 10 only. After biofield treatment both treated cells were analysed for its antimicrobial sensitivity, minimum inhibitory concentration value, biochemical reactions and biotype number with respect to control (Gr. I). Results: The antimicrobial susceptibility and minimum inhibitory concentration of S. aureus showed significant (86.67%) alteration in lyophilized cells while no alteration was found in revived treated cells as compared to control. It was observed that overall 37.93% (eleven out of twenty nine) biochemical reactions were altered in the treated groups with respect to control. Moreover, biotype numbers were substantially changed in revived treated cells, Gr. II (303137, Staphylococcus capitis subsp. ureolyticus) on day 5 and in lyophilized treated cells, Gr. III (767177, S. cohnii subsp. urealyticum) on day 10 as compared to control (307016, S. aureus). Conclusion: The result suggested that biofield treatment has significant impact on S. aureus in lyophilized treated cells with respect to antimicrobial susceptibility, MIC values and biochemical reactions pattern. Apart from these, biotype numbers with new species were observed in revived treated group on day 5 as Staphylococcus capitis subsp. ureolyticus and in lyophilized cells as Staphylococcus cohnii subsp. urealyticum with respect to control, i.e., S. aureus.