Fifteen new aliphatic metabolites, 2-methylpyrimidin-4(3H)-ones (1,2), 2-methoxy-2-methyl-1,2-dihydro-3H-pyrrol-3-ones (4a/4b, 5a/5b), butyrolactones (6-9), and aliphatic metabolites (16-20) as well as known pyridin-2(1H)-one (3) and butyrolactone analogues (10-15) were obtained from the fermentation broth of Streptomyces antifungus isolated from the forest soil sample collected in Tengchong, China. Pyrimidin-4(3H)-one derivatives (1, 2) with an individual 2-methylpyrimidin-4(3H)-one skeleton is a kind of rarely reported compound and were firstly obtained from natural source. The structures of the new metabolites were elucidated by comprehensive spectroscopic analysis including data from experimental and calculated ECD spectra as well as Mosher's reagent derivative method. Compounds 1, 2, 18, and 19 exhibited optimal activity against Staphylococcus aureus with MIC values ranged from 12.5 to 50 μg/mL. Further investigation revealed that 1 effectively inhibited biofilm formation and destroyed the preformed biofilm of S. aureus through oxidative damage, thereby exerting antibacterial effect.