To determine microbial populations and their viability, methods assessing colonies formed and turbidity are generally used. However, these methods require a long incubation time. Thus, a rapid colorimetric microbial viability assay based on the reduction of the tetrazolium salt WST-8 via 2-methyl-1,4-naphthoquinone as an electron mediator was developed. The applicability of the assay for susceptibility testing of various bacteria to antibiotics, screening of antimicrobial substances, determination of water-soluble vitamins, and differentiation of Gram-positive and -negative bacteria was investigated. Good agreement was observed between results with the WST-8 colorimetric method and those obtained using conventional methods. The present assay method was superior to the conventional methods with respect to the overall rapidity, accuracy and convenience.