Fungal diseases are currently a serious public health problem, due to the limited number of really effective principles, and the emergence of resistant strains to the polyenic antifungals. The aim of this study was to screen, for non-polyenic antifungals production by Actinobacteria, and to validate the screening program by characterizing the produced compounds. Actinobacteria isolates were tested against four clinic human pathogenic fungi isolated from Hospital Mohammed V Rabat, Morocco. The production of non-polyenic antifungal metabolites by active isolates was investigated based on the yeast cell specificity as challenging targets, antibacterial activity, activity against resistant Candida tropicalis R2 and Pythium irregular (resistant to polyenes), inhibition of antifungal activity by the addition of exogenous ergosterol, and the UV-visible light spectrophotometric analysis of the active crude extracts. The antifungal compound produced was purified using various chromatographic techniques and the selected producing strain was identified using the polyphasic approach. Among 480 Actinobacteria isolates, 55 showed antifungal activity against all tested clinically derived fungi. After performing the screening program, 4 Actinobacteria that had all the desired criteria were selected. Using the polyphasic approach, the taxonomic position of the selected  Streptomyces AS25, isolated from rhizospheric soil of Alyssum spinosum, showed that it belongs to Streptomyces genus with 100% partial 16S similarity with Streptomyces albidoflavus NBRC13010. On the basis of HPLC and mass spectrometry, the purified compound produced by Streptomyces AS25 was identified as a non-polyenic lactone, antimycin A19, which has been found for the first time to be produced by Streptomyces albidoflavus strain. Following the obtained results, it is important to note that our screening criteria for non-polyenic antifungals have been validated and the rhizospheric soil represent an interesting source to isolate Actinobacteria.