In the framework of exploitation of antimicrobial activity of Actinomycetes in Papua, one hundred isolates of Actinomycetes isolated from soil and leaf litter samples from various ecosystems in Batanta and Salawati Island, Raja Ampat, West Papua were screened. We obtained 200 crude extracts from 100 isolates based on two extraction phases. Nonpolar metabolites were extracted by ethyl acetate : methanol (4:1) solvent while the polar metabolites were concentrated using a freeze-drying method. Based on the agar dilution method, a total of 43 from 200(21.5%) crude extracts have antimicrobial activity against bacteria and yeasts (Escherichia coli NBRC 14237, Bacillus subtilis NBRC 3134, Staphylococcus aureus NBRC 13276, Micrococcus luteus NBRC 1367, Candida albicans NBRC 1594 and Saccharomyces cerevisiae NBRC 10217). Some crude extracts showed anti-Gram negative (1.5%), anti-Gram positive (17%) and antifungal (17%) activities. Crude metabolites which were extracted using ethyl acetate : methanol were more effective on antimicrobial activity (35%) compared with water extraction (17%). Five most potential isolates (BL-13-5, BL-06-5, BL-14-2, BL-22-3, and Sl-36-1) were identified based on 16S rRNA gene sequence data. Sequence similarity search by BLAST program revealed that they show sequence similarities to Streptomyces kanamyceticus (92%), Streptomyces verne (92%), Streptomyces narbonensis (92%), Streptomyces malachitofuscus (98%), and Streptomyces hygroscopicus (96%), respectively.