Healthcare-associated infection (HCAI) is a global health challenge and methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum beta-lactamases (ESBL) producing gram-negative bacilli, which were isolated from HCAIs, are the most common antimicrobial-resistant pathogens. The present research aimed to determine the chemical composition and antimicrobial activity of ethyl acetate and methanol extracts of Nigella sativa seeds. Antimicrobial activity of plant extracts was observed against 20 clinical yeast (which isolated from swab samples of tongue dorsal surfaces in patients with stomatitis), 18 clinical bacteria isolates (which found from HCAIs), and also eight bacteria, and three yeast standard strains by using a broth microdilution assay. The methanol extract had the highest inhibitory activity against S. aureus standard strain at 0.4 mg/mL. The ethyl acetate extract showed the highest antibacterial activity against Bacillus cereus standard strain at 0.4 mg/mL. The methanol and ethyl acetate extracts did not show inhibitory activity against Gram-negative bacteria and yeast isolates. The LC-Q-TOF-MS profiles of the extracts were found to contain saponin, alkaloid, and fatty acid. Mass spectra revealed the presence of 14 fatty acids, four saponins, and three alkaloids for methanol extract and 13 fatty acids, three saponins, and two alkaloids for ethyl acetate extract. © 2022 Marmara University Press.