Rhizosphere soil is one of the unique microbial habitats, especially the actinobacteria. This study was carried out to screen and isolate actinomycetes exhibiting antifungal activity. Initially, the soil samples collected from Allium ascalonicum L., Alpinia galangal, Amorphophallus sp., Clausena harmandiana, and Cymbopogon citratus were pretreated by air-drying and subsequent heat incubation. By using three different media (international Streptomyces project 1 and 2 (ISP-1 and ISP-2) and starch casein agar (SCA)), 79 isolates were obtained and identified as actinomycetes by morphology. Ten actinomycete isolates were then randomly selected to assess their antifungal activity against five pathogenic fungi: Aspergillus sp., Colletotrichum sp., C. acutatum, C. gloeosporioides, and Lasiodiplodia theobromae. Initial screening showed that there were three most active strains, namely CN1, CN4, and LG5, exhibiting a strong and broad spectrum. The bacterial culture supernatants were then prepared and evaluated for their stability under various pH and temperature conditions. The data obtained showed that the antifungal activity of the three culture supernatants was varied depending on the bacterial source and testing fungal strains. The present work implies that rhizospheric soil samples are an attractive source for the discovery of numerous actinomycetes with activity against phytopathogenic fungi. Identification of the bacterial species and determination of the active compounds are essential for further analysis for use in fungal control.