The total content of flavonoids of extracts of Apocynum venetum (AV) fibers were determined by UV spectrophotometer. The antimicrobial activities of AV extracts were also tested using agar plate diffusion method. The results showed that the total content of flavonoids of extracts of AV fibers was 0.76 mg/g. The ethanol extract of AV fibers were partitioned by EtOH, n-BuOH and H2O. Each part was screened for antibacterial activities. The EtOH faction of extract was the most effective against Staphylococcus aureus, Staphyloccus edidernidis, Bacillus lentus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. The fractions of n-BuOH and H2O did not show any inhibitory effect on the tested bacterial and fungi. The present work is the foundation for further isolating and purifying the bioactive components in AV fibers.