Phytochemical composition of ethanol leaf extract of Diodella sarmentosa was profiled with GC-MS and the inhibitory property of the extract against total microbial dehydrogenases were assessed. The major constituents of the extract were squalene (29.50%), Phytol (24.68%), phenol, 3-pentadecyl- (18.58%), 1-Butanol, 3-methyl- (9.09%) and n-Hexadecanoic acid (7.78%). The minimum inhibitory concentration (MIC) of the extract against broad spectrum of microbial population was assessed. Bacillus subtilis, Candidas spp, and Penicillium spp were more sensitive to the treatment and thus; were further investigated using Dehydrogenase activity assay method. Total dehydrogenase activities of Bacillus subtilis, Candidas spp, and Penicillium spp at the extract concentration range of 0 to 2000mg/ml were progressively inhibited at increasing extract concentrations. The threshold inhibitory concentrations (IC50) of the extracts against Candidas spp, Penicillium spp and Bacillus subtilis were 275micro. g/ml, 322 micro. g/ml and 411 micro. g/ml respectively. Our findings suggested the extract as a useful source of antimicrobial phytochemicals for pharmaceutical use.