Extracts of Synadenium glaucescens Pax are reported to contain biologically active compounds against bacterial and viral infections. This study aimed at isolating pure compounds from its bioactive extracts as well as evaluating their antibacterial efficacy. A phytochemical investigation of the root and stem barks involved total maceration in methanol (MeOH). The root barks extract was then partitioned by Vacuum Liquid Chromatography (VLC) in a solvent gradient system to afford three fractions of Hexane (Hex), Ethyl acetate (EtOAc) and finally MeOH for the least, moderate, and most polar compounds respectively. The MeOH extract of stem barks was also partitioned in the same way by using two solvents: dichloromethane (DCM) and MeOH. All fractions were finally dried on a rotary evaporator at < 60 OC of the water bath. Isolation of pure compound from the EtOAc and DCM led to isolation of four compounds namely, hexacosane (G1), β-sitosterol (G2), octacosyl ferulate (G3) and hexacosanoic acid (G4). Their structures were analyzed and confirmed through NMR, GC-MS and in comparison, with literature. Antibacterial assay for G1, G3 and G4 against P. aeruginosa, E. coli, S. aureus, and E. faecalis was achieved by broth serial microdilution. Compounds, G3 demonstrated strong activity against S. aureus (MIC = 0.125 mg/mL) and weak activity against the rest strains (MIC = 2 mg/ mL). Also, the test results indicated G1 had weak activity against all tested strains (MIC = 2 mg/mL or above). While G4 demonstrated a moderate activity (1.0 mg/mL) against E. coli, S. aureus, E. faecalis and weak against P. aeruginosa (MIC > 2 mg/mL). These findings support traditional use and promise for antibacterial drug agents from S. glaucescens Pax.