The insertion of an ester bond into quaternary ammonium salts (QASs) to obtain esterquats offers a promising approach to reducing the environmental impact of these compounds. Monocationic and dicationic esterquats obtained via O-alkylation of a byproduct formed in the sugar production process from sugar beets, namely, glycine betaine, were evaluated as safer substitutes for the currently commercialized compounds. To assess the environmental implications and compare them with existing products, toxicity assessments were conducted using model organisms from different trophic levels, such as terrestrial plants (Centaurea cyanus L. and Sinapis alba L.), crustaceans (Daphnia magna and Artemia franciscana), and algae (Raphidocelis subcapitata). All compounds were established as safe to terrestrial plants, and only a part of them exhibited toxicity toward aquatic life. The toxicity increased with elongation of the alkyl chain to reach the "highly toxic" category according to the recommended acute toxicity rating scale for tetradecyl betainate bromide. Further alkyl extension led to a decrease in toxicity due to the "cut-off" effect. Synthesized dicationic esterquats were found to be an order of magnitude less toxic than their monocationic analogues. Compounds with shorter alkyl chains (