Background: Occupational exposure to cytotoxic drugs can lead to significant health problems. This study was designed to evaluate the risk of 5-fluorouracil (5-FU) contamination for nurses when disconnecting the infusion line of an elastomeric pump from a non-coring needle, by comparing three configurations: the first one using standard Male and Female Luer lock (Group 1), the second one using a needle-free connector (NFC) and a Male Luer lock (Group 2) and the third one using a closed-system transfer device (CSTD), Qimono® (Group 3). Methods: In this in vitro study, 10 elastomeric pumps for each of the three groups were filled with 5-FU and a tissue mimicking the patient’s arm was placed below the connection between the infusion line and the tubing of non-coring needle. After 48 h of infusion, disconnection was performed by a nurse with a wipe soaked in a mix of chlorhexidine and isopropyl alcohol in order to mimic care practices. For each pump, the tissue, the pair of gloves and the wipes used during the disconnection were collected for analysis. Results: Median level of overall 5-FU contamination (gloves + wipes + tissues) in group 3 was significantly lower than in group 1 ( p = 0.018) and group 2 ( p = 0.036). There was no difference between groups 1 and 2. Results per sample type showed no difference in contamination between the three configurations for the gloves as well as for the tissues. Concerning the wipes, the lowest contamination was observed in group 3, representing a mean reduction of 61% compared to group 1 and a mean reduction of 43% compared to group 2. Conclusion: The use of Qimono® appears to significantly reduce 5-FU contamination when disconnecting infusion lines of an elastomeric pump unlike NFC and standard Luer lock.