[Objective] This study was aimed to isolate and identify the actinobacteria with antitumor activity isolated from a soil sample collected from the Ba-yi town, Nyingchi, Tibet, China. [Methods] Actinobacteria were isolated and purified by agar plate dilution method planting in Gause 1 and glucose/asparagines agar media. To test the biological active effects in vitro of the actinobacteria fermentations and its’ crude extracts, we used MTT assay with 5 human tumor cell lines to monitor the antitumor activity, paper-dish method with 7 indicative microorganism strains (bacteria and fungi), and 4 antibiotics to detect the antimicrobial activity and resistance. The polyphasic taxonomies of the antitumor active strains were studied according to morphological, cultural, physiological, biochemical characteristics and 16S rRNA gene sequence analysis. [Results] We isolated 29 actinobacteria strains of which 6 strains with good antitumor activities were obtained (20.7%). All of the crude extracts of the 6 strains had remarkable inhibiting effects against the 5 human tumor cell lines and different degrees of antimicrobial activity against the indicator microorganisms. The crude sample of 4 of them had the inhibition rate of more than 80% on Hela cell line. Only one active strain had good tolerance for 4 kinds of antibiotics. Taxonomic studies showed that all of the 6 antitumor active strains were related to Streptomyces. These 6 strains may be variants of the three known Streptomyces species (Streptomyces galbus, Streptomyces mirabilis, Streptomyces olivochromogenes) respectively. [Conclusion] The actinobacteria in Ba-yi town of Nyingchi are rich in Streptomyces with antitumor activities and may be a potential microbial resource for antitumor drugs.