To investigate the secondary metabolites produced by Streptomyces ardus WS-65090 from soil and their bioactivities.Five teleocidin compounds (1-5) were isolated from the ethyl acetate extract of fermentation culture of S. ardus strain WS-65090 by preparative HPLC.The compounds were elucidated as new teleocidin analogue 14-O-acetylteleocidin B-2 (1),and new naturally occurring teleocidin analogue 14-O-acetylteleocidin B-3 (2),and three known compounds teleocidin B-2,B-3 and A-2 (3-5) by extensive spectroscopic analysis,including HR-MS,1D and 2D NMR,combined with chemical transformation methods and comparison with literature data,etc.Insecticidal activities and cytotoxicity of compounds 1-5 were investigated under laboratory conditions.All compounds showed insecticidal activities against Spodoptera frugiperda,Helicoverpa armigera,Plutella xylostella, and Aphis craccivora with LC₅₀ values from 36.5 to 305.7 μg/mL,and exhibited cytotoxicity against A875,HepG2 and Marc-145 cell lines with IC₅₀ from 3.08 to 18.40 μM.