Our studies examined the structural features responsible for the antifungal activity of 2-ethoxycarbonyl-1,5-benzothiazepine (7a). Three series of 1,5-benzothiazepine derivatives were synthesized and screened for their antifungal activity. The results suggested that the ethoxycarbonyl group at the 2 position and the imine moiety on the seven-membered ring are essential for activity. The most potent of the synthesized analogues (7a, 7b) were further studied by evaluating their cytotoxicity and mode of action (for 7a). The results showed that compounds 7a and 7b were relatively safe for BV2 cells, but compound 7a interfered with Cryptococcus neoformans cell wall integrity by increasing the chitinase activity. Therefore, compound 7a was considered safe as an antifungal agent for animal cells.