Aim To evaluate the antimicrobial potential of Juglomycins isolated from Streptomyces achromogenes E91CS4, an endophyte of Crocus sativus Linn. Methods and Results The extract from E91CS4 displayed significant antimicrobial activity against several pathogens. The endophyte was identified as S. achromogenes on by 16S ribosomal gene analysis. Chemical investigation of the extract led to the isolation of two naphthoquinone antibiotics, Juglomycin A and B. Juglomycin A inhibited several pathogens, with an MIC value of 13 center dot 7 mu g ml(-1), whereas it was most potent against Escherichia coli, Bacillus thuringiensis and Xanthobacter flavus with MIC values of 6 center dot 8, 3 center dot 4 and 6 center dot 8 mu g ml(-1) respectively. It was found to reduce the biofilm formation in E. coli through inhibition of swimming and swarming motilities and downregulation of fimH gene. The alpha-haemolysin-related gene (hlyA) was also downregulated indicating that the compound is also reducing the virulence in E. coli. In vitro time kill kinetics showed efficient bactericidal activity of this compound. Furthermore, Juglomycin A inhibited bacterial transcription/translation in vitro, while also inducing postantibiotic effect in E. coli. Conclusions Juglomycin A is a potential antimicrobial compound against several bacterial pathogens, particularly, E. coli. Significance and Impact of the Study This study showed the promising potential of Juglomycin A as an antimicrobial agent. Efforts should be made to scale up the production of this compound and conduct further studies to explore its efficacy as an antibiotic, using in vivo models.