The present study focuses on the bioassay guided isolation of antimicrobial compounds from Artocarpusaltilis. The hexane, dichloromethane and methanol extracts were obtained from the dried (twigs of A. altilis) by using Soxhlet extraction. The plant crude extracts were evaluated for in vitro antimicrobial activities revealed that twigs part of A. altilis had potent antimicrobial activities, further separation had been done through column chromatography (CC) of dichloromethane and hexane extracts of A.altilis, which were afforded stigmasterol and cycloartenyl acetate respectively, the two compounds exhibited in vitro antimicrobial activity against S. aureus value of (11±0.5mm) and (11.3±0.5mm). Whereas minimum inhibition concentration (MIC) and minimum bactericidal/minimum fungicidal concentration (MBC/MFC) for isolated compounds were ranged from 62.5-500 µg/ml. Their structures were identified by 1H-NMR, 13C-NMR, IR and the spectra obtained were compared with previous data. This study forms a strong basis for declaring A. altilisas antimicrobial potential source to treat pathogenic microorganisms.