- Thailand is the world’ s biggest producer and exporter of natural rubber or “latex”, from Para rubber tree (Hevea brasiliensis). There are many kinds of mold growing on the sheets of rubber which reduce the weight and quality of the rubber sheets and affect the health of farmers and producers. The objective of this study was to isolate and identify fungal contaminant on unsmoked rubber sheets. Later, screening for potential microorganisms was conducted against fungal contaminant. In this study, ninety-three actinomycetes derived from rhizospheric soil of rubber trees were isolated using selective medium, humic acid vitamin (HV) agar, starch casein (SC) agar and oat meal (OM) agar. The morphological and chemotaxonomic study classified and grouped fifty-five isolates into streptomycetes and twenty-two as non-streptomycetes. Sixteen remained unidentified isolates. Moreover, 2 isolates from termite nest soil in rubber plantation were grouped into genus Streptomyces. Twelve samples of rubber sheets were subjected to isolate fungal contaminants. There were thirty-seven fungal isolates from these unsmoked rubber sheets. The most common fungi were identified such as Aspergillus spp. Fusarium spp. and Pennicilium spp. The inhibition of fungal growth on unsmoked rubber sheets by all isolates of actinomycetes were tested by dual culture bioassay method. It was found that twelve isolates showed potential antifungal activity. Strain Streptomyces sp. NKS-3 showed highest activity against fungal contaminant on unsmoked rubber sheets. It was cultivated in five fermentation liquid media and test against fungal contaminants using paper disc agar diffusion assay method. Potato dextrose broth (PDB) and AMHU-5 were the best media for antifungal activity. The minimal inhibitory concentrations (MIC) value of crude ethyl acetate extract of Streptomyces sp. NKS-3 against Penicillium sp. NKF-7 and Aspergillus sp. NKF-9 were 0.156 mg/ml when compaired with Benomyl®. Furthermore, dual culture volatile test of eight isolate volatile producing endophytic fungi showed that Muscodor cinnamomi CMU-Cib 461 had high activity against ten selected fungi contaminant on unsmoked rubber sheets.