The aim of the present study was to evaluate the release of monomers from three bonding systems and to correlate it with their antibacterial effect. Three bonding systems (Optibond FL®, Xeno III® and Clearfil™ Protect Bond) were tested after storage in ethanol 75 % and human saliva. Twenty samples (n = 10/medium) of each bonding material were prepared and polymerized according to the manufacturers' instructions. Each sample was stored in 1 ml of the respective storage medium. The medium was renewed after 24 h, 7 days, and 28 days and was analysed by LC-MS/MS for the release of substances. Additionally, the antibacterial effect of the unpolymerized components of each bonding system and their polymerized mixture was tested using agar disc-diffusion test with Streptococcus mutans. Only HEMA was found to be released. The amount of HEMA detected in the ethanol samples was significantly higher compared to the saliva samples (p < 0.0001). The release of HEMA was as follows: Clearfil™ Protect Bond < Optibond FL® < Xeno III ^®. According to the agar disc-diffusion test, all materials exhibited certain antibacterial activity. The release of HEMA from all tested materials even after storing in human saliva increases the concerns about their toxicity. Their antibacterial effect seems not be due to the release of substances.