Anthocleista djalonensis has many ethnomedicinal claims, one of which is the treatment of infections. The study evaluated the phytochemical, physicochemical profile and antimicrobial properties of the vacuum liquid chromatographic (VLC) sub-fractions of A. Djalonensis leaves. The plant was collected from Umuoji, Idemili North, Anambra State. The plant leaves were identified, authenticated and the herbarium specimen was deposited, with herbarium number PCG/474/A/057. The leaves were dried, pulverized and extracted using cold maceration with methanol, and fractionated successively into n-Hexane, ethyl acetate and butanol. To obtain the ethyl acetate fraction, it was subjected to vacuum liquid chromatography (VLC). The phytochemical and physicochemical analyses were carried out, using standard protocols. The VLC sub-fractions were subjected to HPLC-DAD analysis. The antimicrobial assay of the VLC sub-fractions was carried out using the agar well diffusion assay. The antimicrobial activity of the VLC sub-fractions was tested against three standard clinical bacterial isolates (Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli) and one fungal isolate (Candida albican). Phytochemical screening showed the presence of alkaloids, flavonoids, tannins, proteins, glycosides, saponins, carbohydrates and steroids. The physicochemical evaluation showed that the plant contains a total ash value of 4.55 % w/w, acid insoluble ash value of 6.80 % w/w, water-soluble ash value of 2.65 %w/w, moisture content of 7.90 %w/w and water-soluble extractive value of 1.02 %w/w. The HPLC-DAD analysis of the VLC sub-fractions suggested the presence of isovitexin, septicine, vanillin, 3-methyl-2, 3, 4- pentanetriol, indo-3- carboxylic acid, Cerebroside and bromohexylamide. The VLC sub-fractions had activity against Pseudomonas aeruginosa only, and there was no zone of inhibition for the fungi. The VLC sub-fractions have very little antimicrobial activity, showing antibacterial activity against Pseudomonas aeruginosa only, with no antifungal activity against Candida albican.