Urgent development of alternative on-farm killing methods for poultry is required following the number restrictions placed on the use of traditional manual cervical dislocation by European Legislation (EU 1099/2009). Alternatives must be proven to be humane and, crucially, practical in commercial settings with multiple users. We assessed the performance and reliability of a novel mechanical cervical dislocation device (NMCD) compared to the traditional manual cervical dislocation (MCD) method. NMCD was based on a novel device consisting of a thin supportive glove and two moveable metal finger inserts designed to aid the twisting motion of cervical dislocation. We employed a 2 × 2 factorial design, with a total of eight stockworkers from broiler and layer units (four per farm) each killing 70 birds per method. A successful kill performance was defined as immediate absence of rhythmic breathing and nictitating membrane reflex; a detectable gap in the vertebrae and only one kill attempt (i.e., one stretch and twist motion). The mean stockworker kill performance was significantly higher for MCD (98.4 ± 0.5%) compared to NMCD (81.6 ± 1.8%). However, the MCD technique normally used by the stockworkers (based previous in-house training received) affected the performance of NMCD and was confounded by unit type (broilers), with the majority of broiler stockworkers trained in a non-standard technique, making adaption to the NMCD more difficult. The consistency of trauma induced by the killing methods (based on several post-mortem parameters) was higher with NMCD demonstrated by “gold standard” trauma achieved in 30.2% of birds, compared to 11.4% for MCD (e.g., dislocation higher up the cervical region of the spine i.e., between vertebrae C0–C1, ≥1 carotid arteries severed), suggesting it has the potential to improve welfare at killing. However, the results also suggest that the NMCD method requires further refinement and training optimization in order for it to be acceptable as an alternative across poultry industry, irrespective of previous MCD technique and training.