Ergot alkaloids are novel pharmaceutical and therapeutic agents synthesized in this study using fungal speciesPenicillium citrinum.To get the maximum yield of ergot alkaloids a statistical process of response surface methodology was employed using surface culture fermentation technique. Initially, the strain ofPenicilliumwas improved using physical (ultraviolet (UV) and chemical (ethyl methane sulfonate (EMS) treatments to get the maximum yield of ergot alkaloids through surface culture fermentation technique. After improving the strain, survival rate of colonies ofPenicillium citrinumtreated with UV and EMS was observed. Only 2.04% living colonies were observed after 150 min of exposure ofPenicillium citrinumin UV light and 3.2% living colonies were observed after 20 min of the exposure in EMS. The mutated strains ofPenicillium citrinumwere screened for their production of ergot alkaloids and after fermentation experiments, maximum yield was obtained from PCUV-4 and PCEMS-1 strains. After strain improvement, Plackett-Burman design (PBD) and Box-Behnken design (BBD) of RSM were employed and 10-fold yield enhancement (35.60 mg/100 mL) of ergot alkaloids was achieved. This enhancement in yield of ergot alkaloids proved the positive impacts of RSM and UV on the yield of ergot alkaloids. The study provides a cost effective, economical and sustainable process to produce medically important ergot alkaloids which can be used in various pharmaceutical formulations to treat human diseases.