Exposure of handlers’/operators’ hands is a main route of agricultural pesticides entry into their body. Non-occupational handlers still lack information about appropriate selection of protective gloves to minimize exposure and reduce adverse effects of these chemicals. According to the results of our previous survey, six commercially available, water-resistant gloves commonly used by non-professional gardeners were evaluated for permeation of Acetamiprid, Pirimicarb, and Chlorpyrifos-methyl (Chlorp-m) pesticides by means of in vitro testing. In-use conditions were mimicked as close as possible. Chlorp-m through latex was observed inside the glove from >10 to ≤ 15 min; however, Acetamiprid and Pirimicarb through neoprene/latex and all the three pesticides through butyl were not observed inside gloves for the duration of the experiments (the Breakthrough time (BT)>8 h). The 1-h exposure proved the interior glove contamination with Chlorp-m through disposable latex, vinyl, and nitrile gloves (51, 33, and 41% of applied dose (AD), respectively) just as with Acetamiprid and Pirimicarb through latex glove (11 and 14%AD, respectively). However, when storing the used gloves for 4 days after the exposure, no release of the three pesticides from the butyl and Acetamiprid from neoprene/latex gloves was detected. In all other cases, pesticides were found in the interior glove (36–79, 31–63, and 51–81%AD for Acetamiprid, Pirimicarb, and Chlorp-m, respectively). If used repeatedly, gloves contaminated in this way lose their protective function but give the user a false sense of security. The results suggest that (i) water-resistant gloves are not necessarily pesticide resistant; (ii) disposable latex gloves commonly worn by non-professional gardeners provide inadequate protection even for a short-time contact with pesticides; (iii) to assess the efficiency of reusable gloves, not only BT value but also the reservoir/release effect of parent pesticide and its degradation products should be evaluated; and (iv) awareness-raising activities for non-occupational handlers of pesticides should be enhanced.