Aims Different endophytic fungi were isolated and screened for their digoxin-producing ability. Strain improvement and different culture conditions were studied for more effective production of digoxin. Methods and Results Among the isolated fungi, an isolate produced digoxin in a concentration of 2 center dot 07 mg l(-1). The digoxin-producing fungal isolate was identified as Epicoccum nigrum Link according to the morphological features and phylogenetic analyses. The potentiality of the fungal strain for production enhancement of digoxin was performed by gamma radiation mutagenesis. Gamma irradiation dose of 1000 Gy intensified the digoxin yield by five-fold. Using this dose, a stable mutant strain with improved digoxin productivity was isolated and the stability for digoxin production was followed up across four successive generations. In the effort to increase digoxin magnitude, selection of the proper cultivation medium, addition of some elicitors to the most proper medium and several physical fermentation conditions were tested. Fermentation process carried out in malt extract autolysate medium (pH 6 center dot 5) supplemented by methyl jasmonate and inoculated with 2 ml of 6-day-old culture and incubated at 25 degrees C for 10 days stimulated the highest production of digoxin to attain 50 center dot 14 mg l(-1). Moreover, cytotoxicity of digoxin separated from the fungal culture was tested against five different cancer cell lines. Based on the MTT assay, digoxin inhibited the proliferation of the five different cancer cell lines and the recorded 50% inhibitory concentration ranged from 10 center dot 76 to 35 center dot 14 mu g ml(-1). Conclusions This is the first report on the production and enhancement of digoxin using fungal fermentation as a new and alternate source with high productivity. Significance and Impact of the Study These findings offer new and alternate sources with excellent biotechnological potential for digoxin production by fungal fermentation. Moreover, digoxin proved to be a promising anticancer agent whose anticancer potential should be assessed in prospective cancer therapy.