Isothiocyanates (ITCs) are one of the many classes of breakdown products of glucosinolates found in plants and exhibit biologic activity against various pathogens. In this work, aliphatic isothiocyanates were prepared and the antimicrobial activities against plant pathogenic fungi and bacteria were tested to understand the structure– activity relationships. The results indicated that longerchain derivatives exert a steric inhibition on toxicity of ITCs against Rhizoctonia solani because of steric hindrance and the order of the eight aliphatic ITCs was ethyl[n-propyl[methyl[n-hexyl[n-octyl[n-butyl[ n-heptyl [n-pentyl. Because the hydrophobicity of ITCs was enhanced by increasing alkyl chain length, the antibacterial activity of ITCs against Erwinia carotovora was moderately intense with an increase in hydrophobicity and the order was n-octyl[n-pentyl[n-heptyl [n-hexyl[n-propyl [n-butyl[methyl [ethyl. The present study revealed that some of the compounds exhibited promising antimicrobial activity and could be used as an acceptable alternative to the traditional synthetic fungicides for controlling R. solani and E. carotovora.