Sexuality in fungi has long been a matter of concern and debates that always necessitated extensive analysis of the relationship between organisms assumed to represent different developmental forms of the same organism. Random amplification of polymorphic DNA (RAPD), amino acids, fatty acids and secondary metabolites profiles were performed for four isolatesbelong to ascomycetes fungi; Aspergillus nidulans and Aspergillus chevalieri and their teleomorph Emercilla nidulans and Eurotium chevalieri. Comparison of their activities as antibacterial and antifungal potency against many of Gram positive, Gram negative bacterial and several fungal species including yeast were determinate. Changes in fatty acid, secondary metabolite and RAPD profiles of sexual and their corresponding asexual isolates were observed. Oleic acid was of lower concentrations in A.nidulans and A. chevalieri than in E. nidulans and E. chevalieri while the opposite was observed for linoleic and linolenic acids. RAPD bands of molecular weights of 559 and 790 bp were the only different ones between A. chevalieri and E. chevalieri using primer 5 while those of molecular weights of 1239 and 1757 bp using primer 3 as well as that of 1209 bp using primer 5 represented the only different bands between A. nidulans and E. nidulans. Some intra- and extracellular secondary metabolites were undetected in the imperfect isolates while were detected in the corresponding perfect ones as contract with amino acids percentage detection where exhibited in imperfect isolates much more than perfect one. Deep relationship among cleistothecia formation, amino acids, fatty acid and secondary metabolite biosynthesis has been shown. Study of biological potency of four ascomycetes fungi exhibited great activities of Emercilla nidulans and Eurotium chevalieri (cleistothecia producing isolates) against Gram negative bacterial growth and yeast like fungi on contrast Aspergillus nidulans and Aspergillus chevalieri (two non-cleistothecia producing ones) show high inhibition of growth of Gram positive bacterial and filamentous fungi isolates.