Abstract Rhizosphere microorganisms play an important role in the soil ecosystem. Thirty three different isolates of Streptomyces isolated from healthy tomato rhizosphere soil. Antifungal activities against Colletotrichum acutatum)Accession no. KT215296) were checked by streak method. Isolate TS9 displayed better outcomes against C. acutatum as compared to other isolates. High Performance Liquid Chromatography was used for antifungal compounds from isolate TS9 by comparing with other six isolates that did not showed antifungal activity. The active compound was between (50–60) retention times other peaks did not chosen because the peaks were available in the other isolates which they did not show any effect on the C. acutatum. 10 fractions, named as F1 to F10, came from the preparative HPLC separation of the selected crude extract. One fraction showed highest inhibition to C. acutatum and it was at the retention time (56.232). The selected peak was compared with the 8 standards and was identified as (quercetin) when completely matched with the standard. The present study reports the purification of potential antifungal compound from Streptomyces zaomyceticus isolated from the tomato rhizosphere can be used as a promising fungicide in the near future.