The objective of the current investigation was exploring the influence of extract of Thyme on sperm freezability in buffaloes. Preparation of Thyme ethanolic extract, Evaluation of its antioxidant activity by Diphenyl-1- plcrylhydrazyl (DPPH), High Performance Liquid Chromatography (HPLC) analysis to assess the concentrations of its phenolics and flavonoids. Semen from five mature buffalo bulls was obtained. The main extender was Tris-citric acid- fructose egg yolk (TCFY). Tris diluent with zero Thyme extract was considered as a control. The other extenders are Tris containing ethanolic Thyme extract at concentrations 4, 8, 16 mg/dl as experimental extenders. The semen specimens were supplementary and diluted semen was exposed to the freezing process. Extended semen was estimated for the sperm parameters. The total phenolic content of Ethanolic extract of Thyme (20422.51 µg/g of the extract, mostly Rosmarenic acid, Ferulic acid). The total flavonoids content of Thyme extract (3369.94µg/g of extract, mostly Coffeic acid). The DPPH of Thyme extract (96.5%) with 1000µg/ml and decreased with decreasing concentration. HPLC chromatograms of Thyme extract with peaks of Rosmarenic acid, Ferulic acid and Ferulic acid. Sperm measures were promoted post cooling and thawing in the lower concentration of Thyme (TT₁) if compared to the control and/or the other treatments (TT₂ and TT₃). Enhancement of conception rate was obvious in TT₁ with elevated antioxidants and decreased Malondialdehyde(MDA). It could be fulfilled that, Thymus extract improved post-thawing buffalo sperm quality, the antioxidant status and conception rate (55 %) when used in a low concentration (4 mg /dl ).