Orthosiphon aristatus has been used as a traditional remedy for many diseases, mainly due to its remarkable bioactive phytochemicals. The phytochemicals were extracted with ultrasound energy and then partitioned into individual fractions according to solvent polarity. Two partition techniques, namely liquid-liquid extraction (LLE) and column chromatography (CC), were applied to prepare the fractions from the crude extract. The individual fractions were then subject to LC-MS/MS analysis for compound screening. Rosmarinic acid was the most intense peak and mostly partitioned into ethyl acetate (EA) through LLE, and CC-D from column chromatography. It was also one of the dominant phytochemicals, besides other polar compounds like octadecadienoic acid and its derivatives in radical quenching. However, the non-polar fractions (LLE-EA and CC-B), which contained polymethoxylated flavones, were more effective against the growth of Gram-positive bacteria (Streptococcus agalactiae and Staphylococcus aureus). The Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli) were more susceptible to polar fractions (LLE-AQ and CC-F). Therefore, the polar fractions showed higher antiradical activity, whereas the non-polar fractions appeared to have higher antibacterial activity. Interestingly, O. aristatus extract and its fractions were also better performance than ampicillin in inhibiting bacterial growth, especially E. coli.