Aspergillus is a major human pathogen responsible for the formation of aspergilloma in preformed lung cavity among pulmonary tuberculosis patients. Most of the strains causing Aspergillosis are drug resistant in nature. Hence looking for an effective antagonistic lead compound from natural sources are very much needed to tackle the drug resistance strains. Several antibiotics available in the market are from marine Streptomyces and being explored constantly for newer antibiotics. The aim of the present study was to isolate anti-Aspergillus compound from marine Streptomyces and assay for antifungal activity against drug resistant Aspergillus clinical isolates. In our systematic screening of actinomycetes for anti-Aspergillus activity resulted in isolation of a strain Streptomyces VITSVK5 spp. Bioactivity guided separation yielded a compound 5-(2, 4-dimethylbenzyl) pyrrolidin-2-one (DMBPO) and the anti-Aspergillus activity of the compound was tested against 11 multidrug resistant (MDR) Aspergillus clinical isolates. The compound showed the MIC50 value of 4-0.125μg/ml against MDR Aspergillus clinical isolates by NCCLS method and MIC50 of 3-0.125μg/ml by MTT assay. MTT assay at 50% cell viability was compared with NCCLS method and the former showed 95% agreement with NCCLS method. Based on the results it can be concluded that DMBPO has significant anti-Aspergillus activity against drug resistant clinical isolates.