Bacterial pathogens are the most serious agents causing diseases in both wild and cultured fish resulted in massive mortalities and economic losses. Motile Aeromonas Septicemia (MAS) is a prevalent bacterial disease caused by Aeromonas hydrophila (A. hydrophila) that impacts freshwater fish. This research aimed to evaluate doxycycline (DOX) antibacterial activity against A. hydrophila both in vitro and in vivo. The minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) of DOX against A. hydrophila previously isolated from African catfish (Clarias gariepinus) were determined to be 0.78µg/mL and 3.9μg/mL, respectively. For in vivo experiment, a total number of 80 apparently healthy African catfish, were distributed randomly into four equal groups. Group 1 (non-infected, non-treated) was kept as control, Group 2 (non-infected and treated) was non-infected and treated with 20 mg/Kg BW of DOX for 5 successive days in feed, Group 3 (infected) was inoculated intraperitoneally (IP) with A. hydrophila (2× 10⁸ CFU/ mL) and Group 4 (infected and treated) was infected with A. hydrophila then treated with 20 mg DOX/Kg BW. Our results revealed 70% mortality in African catfish experimentally challenged with A. hydrophila (Group 3). Moreover, significant elevation of serum alanine aminotransferase (ALT) (89±16.26, 54.67±6.44, 36±5.29 U/L, respectively), aspartate aminotransferase (AST) (195±7.64, 221.33±17.9, 211.33±12.72 U/L, respectively) and creatinine (0.68±0.098, 0.76±0.052, 0.58 ±0.023 mg/dL, respectively) was observed on 1^st, 7^th and 14^th days post treatment. While treatment of the infected fish (Group 4) with DOX decreased the mortality rate to 30 %, improved the clinical signs and significantly reduced serum ALT (30.67±6.01, 22.67±1.86 U/L, respectively) and AST (153±7.57, 147.67±6.7 U/L, respectively) on 7^th and 14^th days post treatment. Also, it significantly decreased creatinine (0.21±0.026, 0.25±0.047, 0.21±0.053 mg/dL, respectively) levels at 1^st, 7^th and 14^th days post treatment when compared with those of Group 3. The results showed that DOX could be used as an effective treatment against A. hydrophila infection in African catfish with little adverse effects.