Abstract: Background and Objective: Gardenia (Gardenia jasminoides) has many pharmacological actions such as anti-inflammatory, antioxidant and fibrolytic activities and cytotoxic effects, etc. This study was conducted to recognize the effect of zeatin and thidiazuron (TDZ) on callus proliferation, total phenolic content, total flavonoids and DPPH scavenging activity of gardenia callus cultures. Materials and Methods: Calli were cultured on Murashige and Skoog (MS) medium supplement with different concentrations (2, 4 or 6 mg L–1) of zeatin or TDZ individually as well as combination of 2 mg L–1 zeatin+4 mg L–1 TDZ. Cultures contained 4 mg L–1 TDZ gave the highest callus fresh weight followed by those contained 2 mg L–1 zeatin then that cultured on 4 mg L–1 zeatin. Data reported as Mean±Standard Deviation (SD). Data were subjected to one-way analysis of variance (p< 0.05). Results were processed by Excel (2010) and SPSS Version 17.0. Results: It was found that callus growing on medium supplemented with 4 mg L–1 zeatin gave the maximum value (14.93%) of yield extract. Callus cultured on 4 mg L–1 zeatin recorded the maximum total phenol (268.33 mg GAE/100 g FW of callus) and total flavonoids (2703.33 μg QE/100 g FW of callus) accumulation. The antioxidant activity of each extract was determined through DPPH radical scavenging activity. Callus cultured on 4 mg L–1 TDZ showed the highest antioxidant activity then those cultured on 4 mg L–1 zeatin. The HPLC analysis for phenolic acids showed that chlorogenic acid, rosmarinic acid and cinnamic reached their highest contents with callus cultured on 4 mg L–1 TDZ (123.24, 322.14 and 278.22 μg g–1, respectively). Regarding flavonoids and using HPLC analysis, rutin, apigenin-7-glucoside and kaempferol were detected. Callus cultured with 4 mg L–1 TDZ gave the highest rutin and kaempferol contents (287.76 and 10.38 μg g–1, respectively). However, apigenin-7-glucoside was detected with high content (129.86 μg g–1) in callus culture with 4 mg L–1 Zeatin. Conclusion: The HPLC analysis recommended that TDZ is more effective in accumulation of individual phenolic and flavonoid than Zeatin. The present study provided a useful system for further study on in vitro culture of G. jasminoides as alternative and new source for important secondary products.