With the aim of developing potential antimicrobials, a series of 3,4-disubstituted, 3,4,8 - trisubstituted isocoumarins (3a,b, 5a,b, 10a,b, 13a-f), incorporating nitrogen and oxygen as part of the heteroaromatic ring (piperidine, morpholine, dibenzofuran, coumarin), were synthesized and characterized by IR, 1H NMR, Mass spectroscopy and elemental analysis. In addition their antifungal and antibacterial activities were tested in vitro for their growth inhibitory activities against pathogen fungi, Fusarium pallidoroseum, Chaetonium and against E.Coli, S. Aureus as gram negative and gram positive bacteria. A majority of the compounds were found to be remarkably active against gram negative bacteria. The relationship between the functional group variation and the biological activity of the evaluated compounds is discussed. Antifungal evaluation, in vitro, shows that derivative 6a (Scheme Ib) displayed the highest antifungal and fungicidal activity against Chaetomium and 13b (Scheme III) derivative displayed highest antibacterial activity against gram negative bacteria.