In this study we measured in vitro honey bee enzyme activities in presence of the synergist piperonyl butoxide (PBO) and some new benzodioxole and dihydrobenzofuran derivatives through absorbance and fluorescence spectrophotometric assays, to determine if and to what extent detoxification systems and acetylcholinesterases are affected by these compounds. Both Italian (Italy) and Carniolan (Czech Republic) honey bees were tested. In the case of Italian honey bees the esterase activity was partially but significantly inhibited by almost all the tested products. In Carniolan honey bees only the dihydrobenzofuran derivative EN16-41 significantly inhibited esterases. Other enzymatic systems, such as cytochrome P450 monooxygenases (P450s) and glutathione S-transferases (GSTs), often involved in xenobiotics detoxification, as well as acetylcholinesterases (AchEs) seem not to be targeted by the studied synergists. These data seem to suggest that some of the investigated PBO analogues could not be detrimental to honey bees.