A new naturally occurring sterol, compound 5, and six known stigmasterols were isolated from fruits of Ailanthus altissima Swingle by repeated column chromatography and RP-HPLC. Their structures were identified as, 5${alpha}$-stigmastane-3,6-dione (1), 3${eta}$-hydroxystigmast-5-en-7-one (2), stigmast-5-ene-3${eta}$, 7${alpha}$-diol (3), 6${alpha}$-hydroxystigmast-4-en-3-one (4), 5${alpha}$-stigmastane-3${eta}$, 6${eta}$-diol (5), stigmast-4-ene-3${eta}$, 6${alpha}$-diol (6), stigmast-5-ene-3${eta}$, 7${alpha}$, 20$xi$-triol (7) by spectral analysis and comparison with the published data. These compounds have not been reported from genus Ailanthus, whereas compound 7 was identified by NMR for the first time. In addition, the $95\%$ ethanol extract and compounds from the fruits of Ailanthus altissima SWINGLE were assayed for in vitro antimicrobial activity. The extract was potent active against the assayed bacteria while compounds 3 and 7 exhibited moderate activity.