Abstract: Photopharmacology is one of the most promising and rapidly developing branches of modern medicine, based on the control of the biological activity of compounds by the light effect. An urgent issue in the development of “smart drugs” (compounds with light-controlled biological activity) is the toxicological assessment of new synthesized substances, determining their safety for humans and animals. This study is related to investigation of toxic properties of vinyl phosphonates, diene compounds with ethyl and isopropyl substituents at the phosphonate group. To determine the toxicity, test organisms from various systematic groups have been used, such as daphnia, ciliates, and green unicellular algae, which makes it possible to obtain the most objective assessment of the considered substances. The performed study of the phosphonates has revealed the differences in the degree of their toxicity for the test organisms. Daphnia have turned out to be the most sensitive to the action of functionalized phosphonates, whereas the unicellular green algae have exhibited slightly lower sensitivity, and the ciliates have shown the least sensitivity to the synthesized agents. Based on experiments on daphnia, considered as the most sensitive test organisms, median lethal dose and and safe concentration of the phosphonates have been determined.