The speed of efficacy of afoxolaner (NexGard®) against Ctenocephalides felis fleas was evaluated in two studies. Study A assessed the efficacy against existing fleas whereas study B assessed the efficacy against new infesting fleas. In study A, 12 dogs were allocated to the untreated group and 20 dogs to the treated group. All dogs were infested by 100 fleas each at Day −1, treated at Day 0 and flea combed at 2 h or at 6 h post treatment. In study B, 6 dogs were allocated to the untreated group and 10 to the treated group. They were infested with 100 fleas each on Days 2, 7, 14, 21 and 28. Fleas were removed and counted at 6 h post-infestation. Immediate and persistent efficacies were evaluated by counting fleas on the dogs. To evaluate induced mortality after exposure on dogs, fleas collected alive were placed in an insectarium for 24 h and assessed for viability. The immediate efficacy on dogs was significant at 6 h with 100%. The induced death of the fleas collected live from dogs 2 h after exposure was 99.7%. Concerning new infesting fleas, the observed efficacy at 6 h and the induced mortality were significantly different (p < 0.05) from the control at all time-points. At 6 h, the prophylactic efficacy was > 97% at Day 2 and Day 8 and > 90% at Day 14. The induced mortality after 6 h of exposure on dogs varied between 73.3% and 100% for the whole study.