The goal of this study was to utilize gas chromatography-mass spectrometry to characterize the bioactive phytochemicals found in the petroleum ether fraction of Gardenia thunbergia Thunb. leaves methanol extract. The GC-MS analysis yielded several peaks, indicating the existence of 23 compounds tentatively with various medicinal properties. Palmitic acid, butyl palmitate, acetyl tributyl citrate, methyl palmitate, cholesta-4,6-dien-3-ol, and ursolic acid methyl ester were found to be the most prevalent compounds. In this study, the antibacterial effect of total methanol extract and petroleum ether fraction against Staphylococcus aureus was evaluated and compared with the positive control (vancomycin) and the negative control (dimethyl sulfoxide). Twenty-two S. aureus clinical isolates were obtained for the experiment. The values of MICs were detected, and the effect of the tested extracts on the biofilm formation ability of S. aureus isolates was evaluated. The total methanol extract and the petroleum ether fraction have notable antibacterial activity against the tested S. aureus clinical isolates.