The antimicrobial potential of two bioflavonoids, i.e., 5,7-dihydroxy-4',6,8-trimethoxyflavone (1) and 5,6-dihydroxy-4',7,8-trimethoxyflavone (2), isolated from Limnophila heterophylla Benth. and L. indica (Linn.) Druce (Scrophulariaceae), respectively, were evaluated against the microbial strains Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Alternaria solani, and Candida albicans. Compounds 1 and 2 exhibited moderate but broad antimicrobial activities against both Gram-positive and Gram-negative bacteria and also against the fungal pathogens. Moreover, the mechanism of action of 1 and 2 on the cellular functions or structures of some of the microorganisms was studied. Compound 1 showed a bactericidal effect against E. coli and S. aureus (MICs of 200 and 250 μg/ml, resp.), while compound 2 was found to effectively kill B. subtilis by cell lysis. The growth of A. solani and C. albicans was inhibited by compounds 1 and 2, respectively. The effects of the flavonoids on the cellular structures and the carbohydrate metabolic pathways were studied by scanning electron microscopy (SEM) of the treated cells and by assessing the specific activity of key enzymes of the pathways, respectively. At sublethal doses, they enhanced the activity of gluconeogenic fructose bisphosphatase, but decreased the activity of phosphofructokinase and isocitrate dehydrogenase, the key enzymes of the EmbdenMeyerhofParnas pathway and the tricarboxylic acid cycle, respectively.