Background: There is limited evidence on the use of video laryngoscopy (VL) in neonatal tracheal intubation (NTI) during neonatal resuscitation. In this study, we aimed to compare the difference between direct laryngoscopy (DL) and VL in NTI of trainees during neonatal resuscitation training. Materials and Methods: A prospective observational study was conducted during a neonatal resuscitation training course to examine three circumstances: NTI by experienced medical staff (EMS) and less-experienced medical staff (LEMS) in a neonatal resuscitation scenario; NTI by EMS and LEMS with an ongoing chest compression; and NTI by midwives who were novices in the procedure. The trainees were given scenarios or were shown demonstrations on newborn simulation manikins and were required to perform an NTI on a simulation manikin using DL and/or VL. The mean intubation time and success rate of intubation were measured. Results: The mean NTI time for EMS using VL (24.1 ± 7.2 s) was significantly longer than that using DL intubation (18.1 ± 6.9 s, P < 0.001), whereas there was no significant difference between using VL and DL for LEMS. EMS spent slightly less time on NTI than did LEMS using both VL and DL, but there were no statistically significant differences (both p > 0.05). The NTI success rate for EMS using VL (48.0%, 12/25) was significantly lower than that using DL (88.0%, 22/25, P = 0.004), while the NTI success rate for LEMS using VL (68.2%, 15/22 vs. 40.9%, 9/22) was higher than that using DL, but there was no statistical significance. When NTI was required with ongoing chest compressions, there was no significant difference in the mean NTI time and success rate between using VL and DL for EMS or LEMS. In the group of midwives who were novices in NTI, after they watched a demonstration teaching NTI, the intubation time using VL (19.6 ± 9.0 s) was significantly shorter than that using DL (28.0 ± 6.7 s, P < 0.001). The success rate of NTI using VL was significantly higher (96.2%; 25/26) than that using DL (69.2%; 18/26). Conclusion: The video laryngoscopy could be an effective training tool for inexperienced staff in developing the skill of tracheal intubation.