The methanolic crude and methanol–aqueous extract of Alstonia macrophylla leaves and n-butanol part of the crude extract showed antimicrobial activity against various strains of Staphylococcus aureus, Staphylococcus saprophyticus, Streptococcus faecalis, Escherichia coli, Proteus mirabilis, Trichophyton rubrum, Trichophyton mentagrophytes var. mentagrophytes and Microsporum gypseum. The minimum inhibitory concentration (MIC) values ranges from 64 to 1000 μg/ml for bacteria and 32–128 mg/ml for dermatophytes. However, the strains of Pseudomonas aeruginosa, Klebsiella sp. and Vibrio cholerae showed resistance against in vitro treatment of the extracts up to 2000 μg/ml concentration, while the two yeast species were resistant even at 128 mg/ml concentration. The stem bark extract prepared similarly was found to be less active compared to the leaves. Phytochemical study indicates that the crude extract contains tannins, flavonoids, saponins, sterols, triterpene and reducing sugars. Further fractionation and purification of n-butanol part of the extract showed the presence of β-sitosterol, ursolic acid, β-sitosterol glucoside and a mixture of minor compounds only detected in TLC.