Combination therapy of antibiotics is leading to improved efficacy or safety profiles with decrease emergence of bacterial resistance. Because of this benefit, many of antibacterial combinations have been used in veterinary practice for the past few decades. The purpose of this study was to examine the in vitro activity of an amoxicillin alone and in combination with other antibiotics against pathogenic bacteria of fish origin. Based on the fractional inhibitory concentration (FIC) index (FIC less-than or equal to 0.5), a synergistic interaction was shown in combination of florfenicol with amoxicillin or cefuroxime. The combination of florfenicol and amoxillin showed higher antibacterial activity than that of florfenicol and cefuroxime. Ratio of amoxicillin and florfenicol in combination was 1 : 1, which showed the antibacterial activity against bacterial isolates of fish as compared with other ratios. A synergetic effect of the combination (amoxicillin and florfenicol) was further confirmed in the time-kill curve study. The study showed a better in vitro antibacterial activity of a 1 : 1 combination of amoxicillin and florfenicol than the individual antibacterial against bacterial isolates of fish. In conclusion, the combination of florfenicol and amoxicillin may serve as a potential antibacterial therapy in fishes infected pathogenic bacteria.