Endophytes are either bacteria or fungi that reside in the tissues of the plant without causing any apparent symptoms. Some endophytic microorganism may promote growth of plants, help in uptake of nutrients and increase the ability to bear environmental stresses, like salinity, drought, and reduce biotic stresses. During our study, plants were collected from different localities in Karachi Pakistan, like Memon Goth, Kathor, Gadap, Gharo, Malir and University of Karachi campus from which endophytic Penicillium were isolated. Out of the eightysamples of the plant, 14isolates of endophytic Penicillium isolated, (root, stem and leaves) from wild plants (Achyranthus aspera, Atriplex stocksii, Euphorbia hirta, Chorchorus tridens) and cultivated plant (Solanum melongena, Lycopersicon esculentum, Helianthus annuus, Azadirachta indica, Abelmoschus esculentus, Momordica charantia) Species of Penicillium identified as P. asperum, P. lilacinum, P. purpurogenum, P. nigricans, P. rugulosum, P. restrictum, P. duclauxi, P. citrinum, P. thomii, P. lividum and P. javanicum. Identification of selected isolates of Penicillium was also confirmed by using molecular biology tools. Antimicrobial activity of 14 endophytic isolates of different species of Penicillium tested against common fungi (root rotting) viz., F. oxysporum,, Fusarium solani, Macrophomina phaseolina and Rhizoctonia solani by dual culture plate assay. All EP isolates showed significant resultproduced by the inhibition zone. Nematicidal potential of cell free culture filtrates of endophytic Penicillium also has shown significant results. After 24 hour 50%nematicidal potential showed by P.purpurogenum (EP-3) while after 48 hours all other isolates showed 100% mortality. Culture filterates of endophytic Penicillium causedgrowth suppression ofbacteria Salmonella typhimurium, Bacillus subtilis,Escherichia coli and Staphylococcus aureus. Asconcentration increased biocontrol potential of culture filterates of EP increased as well. These outcomes show that endophytic Penicillium could be fullfil the need of discovering of new antibiotics. Culture filtrates of Penicillium also showed activity of fungicidal against root rotting fungal pathogens F.solani, R.solani, M.phaseolina, R.solani and F.oxysporum by making inhibitory zone. Cuture filterates of 60 µl/disc showed more effective results than 20 or 40 µl/disc. Fractionation of cell free culture filtrates of viable isolates of our Penicillium (EP), was made in solvents i.e chloroform and n-hexane and showed strong antibacterial and antifungal activity against above described pathogens. These results showed that secondry metabolites having compounds with strong antimicrobial potential. Secondary metabolites producing from endophytic Penicillium spp., offer an stimulating area of investigation for the encounter of novel antimicrobial compounds. Hexane fraction of mycelium of promising isolate EP-5 showed prescence of chemicals. In current research antagonistic potential of Penicillium was assessed against phytopathogens on sunflower (Helianthus annuus), chickpea (Cicer arietinum), tomato (Lycopersicon escolentum), mungbean (Vigna radiata) and okra (Abelmoschus esculentus) in field and screen house experiments. Inhibitory affects on F.oxysporum, R.solani, F.solani, and M.phaseolina showed by many endophytic Penicillium which causes healthy plant growth by improving plant length, fresh shoot weights in both type of experiments (Screen house and field). In some experiment, polyphenol and antioxidant activity also showed significant result which might be due to resistance produced by endophytes. Endophytic Penicillium treated plants produced fruits which is better in quality as compared to control.Endophytic Penicillium associated with healthy plants is a source of new bioactive metabolites, which could be exploited in plant protection and also in medicine.