An efficient methodology to obtain novel antifungal analogs of brassinin 1 is described. Starting from l-tryptophan 2, N,N'-dialkylthiourea 4, 4-[(1H-indol-3-yl)methylene]-2-sulfanylidene-1,3-thiazolidin-5-one 5 and alkyl (2S)-3-(1H-indol-3-yl)-2-{[(alkylsulfanyl)carbonothioyl]amino}propanoate 6 type compounds were obtained as main products in different ratios depending on the reaction conditions via a tandem dithiocarbamate formation and Michael addition reaction. In order to understand the dependence of the reaction conditions on the mechanism pathway, a DFT/B3LYP study was performed. The results suggested the existence of competitive mechanistic routes which involve the presence of an ionic dithiocarbamate intermediate 9. Antifungal activities of all products were then evaluated against Fusarium oxysporum through mycelial growth inhibition using a microscale amended-medium assay. IC50 values were thus determined for each compound. These results showed that 6-related compounds can be considered as promissory antifungal agents.