In this study, we used efficient, selective methods to systematically isolate actinomycetes from 23 soil samples collected from Kanas National Nature Reserve, Xinjiang, China, and obtained 246 strains. Morphological observation and cell wall amino acids analysis indicated that they belonged to seven genera, in which streptomyces was the dominant genus, accounting for 82.9% of the total. Among the different soil pretreatment methods, 0.05% SDS plus 6% yeast extract is the best, using which, we isolated 62.6% of the obtained actinomycetes only on TPA. Activity screening using six commonly used plant pathogenic fungi and TMV found that 6.1% strains exhibited inhibitory activity greater than 80% to at least one of the six fungi and 5.7% strains could inactivate TMV in vitro to certain degrees. The results from this study provide evidence that the actinomycetes in Kanas soils could be promising sources for antimicrobial bioactive agents.