In wastewater treatment plants the antagonistic activities of actinomycetes could be contributing signiﬁcantly in microbe-removing mechanisms, which are a combination of different factors. For this purpose, out of 58 actinomycetes isolates from a wastewater treatment plant in Al-Fayoum Governorate, Egypt, only 36 different morphological isolates were selected for further studies. Although 35 isolates (97.23%) were active against one or more of tested bacteria, WD5 isolate had broad-spectrum antagonistic activity against both Gram-negative and Gram-positive tested bacteria. WD5 ethyl acetate extract recorded the highest antibacterial activity against Staphylococcus aureus MTCC 96 (23 mm) and the lowest antibacterial activity against Pseudomonas aeruginosa MTCC 2453 (11 mm). Gas chromatography-mass spectrometry analysis of extract using available NIST (National Institute of Standards and Technology) library data identified seven bioactive compounds: 2-methylbutyl acetate, 3′,5′-dinitrobenzoic acid, 1-octadecene, tetradecane, dodecane, docosane, and methylamine,N,N-bis(N.-decyl). Many earlier studies mentioned the antibacterial activity of all above seven compounds. The 16S rRNA sequence of WD5 was recorded in GenBank under the accession number MK491056 and confirmed by comparing it with the known 16S rRNA sequences in GenBank as Streptomyces sp. These results indicated antagonistic activity of actinomycetes in the removal of wastewater-associated bacteria.