Three known compounds, stemofoline, (2'S)-hydroxystemofoline, and (11Z)-1 ',2'-didehydrostemofoline, along with two new alkaloids, stemaphylline and stemaphylline-N-oxide, have been isolated from a root extract of Stemona aphylla. The structures of these alkaloids were determined on the basis of their spectroscopic data. The analysis of the crude dichloromethane extract by GC-MS in the ElMS mode showed the presence of alkaloids 1-4, the alkaloid 11, and stilbostemin R. The crude dichloromethane extract and 4 were tested for their comparative biological activities. The results of their acetylcholinesterase (AChE) inhibitory activities showed that the crude extract had higher activity than that of 4. The insecticidal properties of the crude extract and 4, using a topical application, showed that 4 had an activity similar to the positive control, methomyl, whereas the crude extract had much lower activity. Their antimicrobial activity against Escherichia coli ATCC 25922, Staphylococclls allreliS ATCC 25923, Pseudomonas aUl'lIginosa ATCC 27853, and Candida albicans ATCC 90028 was weak (MIC 62.5-125 /tg/mL, MBC 125-250 Itg/mL, MFC 125 !tg/mL) but much higher than that of the crude extract.