The development of functional as well as nutritional surfactants for the food industry remains a matter of great interest. In the present study, a series of 6″-O-acylmaltotriose monoesters bearing alkyl side chains of 10–18 carbons was prepared by enzymatic means. The emulsions derived from those monoesters incorporating palmitoyl, stearoyl, and oleoyl side chains generally displayed advantageous shelf-lives, superior resistance to environmental variations, and more favorable droplet size distributions as well as stronger cytotoxic effects against various cancer cell lines. Ester 6 was shown to significantly inhibit the proliferation of MCF-7 breast cancer cells by inducing G1 phase arrest. Specifically, the levels of the G1 phase-related markers cyclin D1 and cyclin E as well as the cycle-dependent kinase 4 were suppressed by this particular ester. This study thus reveals that maltotriose esters can not only serve as novel functional food emulsifiers but also act, in vitro, as notable cytotoxic agents through a well-defined mechanism-of-action.