This study explores the phytochemical composition, thin-layer chromatography (TLC) profiles, and antibacterial activity of Usnea orientalis motyka extracts using various solvents: acetone, petroleum ether, chloroform, and methanol. Phytochemical analysis reveals that acetone is the most versatile solvent, extracting a wide range of compounds including alkaloids, saponins, glycosides, flavonoids, triterpenes, and steroids. Methanol also extracts multiple phytochemicals but is less comprehensive than acetone. Petroleum ether predominantly extracts alkaloids and glycosides, while chloroform selectively extracts proteins and tannins. Carbohydrates were not detected in any of the extracts. TLC analysis indicates that acetone extracts show the highest mobility of compounds with an Rf value of 0.64, reflecting a strong affinity for the mobile phase. Methanol extracts have the highest Rf value of 0.78, demonstrating the greatest mobility, whereas petroleum ether and chloroform extracts exhibit lower Rf values of 0.54 and 0.71, respectively. Antibacterial sensitivity tests show that gentamicin and piperacillin effectively inhibit Staphylococcus aureus and Pseudomonas aeruginosa, respectively. Gentamicin also inhibits Klebsiella pneumoniae, though to a lesser extent, while ampicillin is effective against Escherichia coli. The DMSO control confirms that the observed effects are attributable to the antibiotics used. Among the Usnea orientalis extracts, the acetone extract exhibits notable dose-dependent antibacterial activity against Pseudomonas aeruginosa, with inhibition zones increasing from 22 mm at 25 µl to 40 mm at 100 µl. The methanol extract maintains stable antibacterial activity with minimal variation in inhibition zone across concentrations, while the hydro methanol extract shows no antibacterial activity.