Formalin, the aqueous solution of formaldehyde, is used as a standard earthworm expellant. Since the possible biochemical effects of formalin to earthworms were not investigated, in the present study adult individuals of the earthworm species Eisenia andrei were exposed to sub-lethal concentrations of formalin in order to determine whether its usage as an expellant will influence the physiological status of the earthworms. In all experiments filter paper contact test was used and experiments were conducted under controlled laboratory conditions. Earthworms were exposed to 0.005, 0.01, 0.05, 0.1 and 0.2 mg ml−1 of formalin for 5 min, 15 min, 30 min and 2 h, and the acetylcholinesterase (AChE) activity, catalase (CAT) activity and concentration of oximes were measured. As expected, the lowest AChE activity was measured at the highest concentration of formalin applied (0.2 mg ml−1). However, following a 2 h exposure to concentration of 0.01 mg ml−1, the AChE activity increased up to 1.12 times the activity in the control. Similar results were obtained when concentration of oximes was measured: the lowest concentration of oximes occurred following 2 h exposure to the highest concentration (0.2 mg ml−1); and the highest concentration of oximes—equating to 1.18 times increase relative to the control—occurred after a 2 h exposure at 0.01 mg ml−1. Dose–response curves for AChE activity showed an inverted U-shape characteristic for hormesis and concentration of oximes indicates a role in maintaining the normal AChE activity in the organism. Measurement of CAT activity measurement showed dose and time dependent induction, indicating the occurrence of oxidative stress. The obtained results showed formalin causes measurable effects on the metabolism of E. andrei, therefore the usage of formalin as an earthworm expellant is unsuitable for ecotoxicological research or biomonitoring.