The most common bacterial diseases in pond-raised channel catfish Ictalurus punctatus (Rafinesque) are enteric septicemia of catfish and columnaris, caused by Edwardsiella ictaluri and Flavobacterium columnare respectively. Medicated feed containing antibiotics is one management approach that catfish producers use in the treatment of bacterial diseases. However, the future use of all types of medicated feed in catfish aquaculture is uncertain. To discover effective alternatives to antibiotics, a rapid 96-well microplate bioassay utilizing E. ictaluri and F. columnare to evaluate natural compounds and extracts was developed. In this bioassay, bacterial growth is determined by absorbance measurements of microplate wells after 24 h incubation and then confirmed by detecting cell viability after the addition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide with additional incubation for 24 h. The minimum inhibitory concentration, minimum bactericidal concentration and 50% inhibition concentration (IC50) are determined by graphing the absorbance data. The 24 h IC50 results of test compounds are compared with the 24 h IC50 results of the drug controls oxytetracycline and florfenicol. Among the antibiotics evaluated, doxycycline and tetracycline appear more effective against E. ictaluri and F. columnare than either drug control. This bioassay is rapid, reproducible and economical for evaluating a large number of compounds and extracts.