Abstract Objectives The aim of this study was to investigate the preventive effect of thymol in in vivo muscle inflammation and regeneration on cardiotoxin-induced injury. Methods Mice were pretreated (p.o.) with thymol (10–100 mg/kg), and after 1 h, cardiotoxin (25 μM, 40 μl) was administrated into the gastrocnemius muscle. The quantification of the areas of inflammation and regeneration of muscle tissue (3, 7 and 10 days) in HE-stained slides as well as the count of total mast cells and different phenotypes of mast cells were made. Sirius red staining was used to analyse total collagen expression. Key findings The pretreatment with thymol significantly reduced the area of inflammation (30 and 100 mg/kg) and increased the area of regeneration (100 mg/kg) 3 days after the cardiotoxin injection. Thymol at 30 and 100 mg/kg increased the area of collagen in 3 days and also decreased this area in 7 and 10 days, compared to the injured group. The pretreatment with thymol did not affect the number of total mast cells; however, it was able to change the number of mucosal mast cells within 10 days. Conclusions This study suggests that thymol ameliorates inflammatory response and accelerates regeneration in cardiotoxin-induced muscle injury.