Four series of dicephalic cationic surfactants, considered as new antielectrostatic agents have been investigated in order to establish their toxicity and biodegradability. Among them N,N-bis[3,3′-(dimethylamine)propyl]alkylamides, N,N-bis[3,3′-(dimethylamine)propyl]alkylamide dihydrochlorides, N,N-bis[3,3′-(trimethylammonio)propyl]alkylamide dibromides and N,N-bis[3,3′-(trimethylammonio)propyl]alkylamide dimethylsulphates with different hydrophobic chain length (n-C9H19 to n-C15H31) and type of counterion (chloride, bromide and methylsulfate) have been studied. The inhibitory effect against microorganisms has been examined using model gram-positive and gram-negative bacteria, and yeasts. None of the tested surfactants have shown antimicrobial activity against gram-negative bacteria (Escherichia coli, Pseudomonas putida) and yeasts (Saccharomyces cerevisiae, Rhodotorula glutinis) at a concentration below 1000μgmL−1, however some of them were moderately active against gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis). The Microtox® test was successfully applied to measure EC50 values of the studied dicephalic cationic surfactants. Their toxicity to Vibrio fischeri depended upon the alkanoyl chain length with the EC50 values in a range of 2.6–980mgL−1. N,N-bis[3,3′-(dimethylamine)propyl]alkylamide dihydrochlorides 2a–b and N,N-bis[3,3′-(trimethylammonio)propyl]alkylamide dibromides 3a–b comprising n-decanoyl and n-dodecanoyl hydrophobic tails appeared to be the least toxic. Furthermore, the biodegradability under aerobic conditions of 2a–b, 3a–b was evaluated using OECD Method 301F. According to the obtained results 2a, 3a–3b can be considered as almost readily biodegradable and they are not expected to be persistent in the environment. Additionally, partial biodegradation was observed for 2b, indicating its possible biodegradation in wastewater treatment systems.