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Background: Botulinum toxin (BoNT) or Botox injections are popular non-surgical and non-invasive option to optimize and change an individual’s facial appearance and achieve rejuvenation. Pyridostigmine is an acetylcholinesterase inhibitor important in clinical practice. This study evaluated the toxic effect of BoNT to demonstrate the efficacy of anticholinesterase inhibitor pyridostigmine. Methods: We conducted an experimental interventional study to evaluate the antidote effect of pyridostigmine against BoNT. Forty rabbits (skeletally mature males, body weight 1000–2000 g) were included in the study. The rabbits were injected with Clostridium botulinum type A neurotoxin complex (BoNT or Botox), which was reconstituted with 0.9% sterile sodium chloride to a concentration of 100 U/2.5 mL. Animals were divided randomly into four groups (10 in each) as follows: Group 1 (control group), group 2 (pyridostigmine-treated group), group 3 (BoNT only group), and group 4 (BoNT + pyridostigmine treated group). Results: In the control group, there was statistical difference between the rabbits’ weight (P=0.03). The left muscle was significantly heavier than the right (P=0.025). In the pyridostigmine only group, the rabbits’ weight had a high statistical difference during four weeks of the study (P=0.002). In the BoNT injection group, weight had high statistically significant difference four weeks of the study (P=0.033), which was more in first week (1.55±0.2 kg) and then subsequently decreased in the fourth week (1.345±0.13 kg). The mean weight of the rabbits’ right quadriceps muscle was 6.573±1.3 g, which was significantly lower than the mean weight of the rabbits’ left quadriceps muscle (8.09±1.2 g, P=0.0001). Conclusions: To the best of our knowledge, this is the first study conducted in Iraq to investigate the antidote effect of pyridostigmine against BoNT injections. Pyridostigmine and saline caused increased weight of rabbits in comparison to rabbits that received BoNT injections. Pyridostigmine can act as strong antidote against Botox toxicity.