Objective: To explore various unexplored locations where Penicillium spp. would be available and study the production of penicillin from the isolated Penicillium spp. in different media with altered carbohydrate source. Methods: The collected soil samples were screened for the isolation of Penicillium chrysogenum (P. chrysogenum) by soil dilution plate. The isolated Penicillium species were further grown in different production media with changes in the carbohydrate source. The extracted penicillin from various isolates was analyzed by HPlC for the efficacy of the product. Further the products were screened with various bacterial species including methicillin resistant Staphylococcus aureus (MRSA). And the work was extended to find the possible action on MRSA, along with characterization using other pathogens. Results: From the various soil and citrus samples used for analysis, only the soil sample from Govment General Hospital of Bangalore, India, and Sanjay Gandhi Hospital, Bangalore, India, showed some potential growth of the desired fungi P. chrysogenum. Different production media showed varied range of growth ofPenicillium. Optimum production of penicillin was obtained in maltose which proved maximum zone of inhibition during assay. Characterization of penicillin on pathogens, like wild Escherichiacoli strain, Klebsiella spp., and MRSA, gave quite interesting results such as no activity on the later strain as it is resistant. HPlC data provided the analytical and confirmation details of the penicillin produced. Accordingly, the penicillin produced from the soil sample of Govment General Hospital had the high milli absorbance unit of 441.5 mAu compared with that of the penicillin produced from Sanjay Gandhi Hospital sample, 85.52 mAu. Therefore, there was a considerable change in quantity of the penicillin produced from both the samples. Conclusions:The Penicillium spp. could be possibly rich in hospital contaminants and its environments. This research focuses on various unexplored sources of medical ailments, and also shows that the growth of penicillin is high in maltose rich media that could possibly enhance the growth.