Anthracnose is one of the most important postharvest diseases of many economically important crops worldwide. This study was conducted with the objective of investigating the sensitivity of Colletotrichum gloeosporioides and C. truncatum isolates to multiple fungicides with different modes of action. The study analyzed quantitative sensitivity data derived from conventional amended agar (AA) assays and qualitative spore responses obtained from a novel microtiter bioassay that is based on reduction of a viability dye, Alamar blue (AB). Generally, for AA assays, the percent growth inhibition (%RGI) increased with increasing concentration for all isolates and all fungicides, except for copper hydroxide. C. truncatum isolates reacted differently to increasing concentrations of the various fungicides depending on whether the isolates originated from pepper or papaya. C. truncatum from pepper had generally less %RGI than C. truncatum isolates from papaya. C. gloeosporioides isolates from papaya had generally higher %RGI than C. truncatum isolates for all concentrations tested for pyraclostrobin, chlorothalonil, and fosetyl-aluminum. C. gloeosporioides isolates from pepper had generally higher %RGI than C. truncatum isolates for all concentrations tested for most fungicides. In all cases, Colletotrichum sp. and fungicide had significant (P ≤ 0.001) effects on the log concentration of fungicide for which relative growth was inhibited by 50 and 90% (log EC50 and log EC90, respectively) calculated for all isolates, regardless of whether values were compared for only C. gloeosporioides isolates or only C. truncatum isolates. Correlation analyses of log EC50 and log EC90 values of all the isolates revealed a nonsignificant association for pyraclostrobin. In AB assays, all fungicides had an equivalent effect at inhibiting spore germination at the lower concentrations. According to binary logistic regression analyses, species, isolate, and fungicide concentration had significant predictive value in determining whether an AB test would be positive. Sequence alignments between C. gloeosporioides isolates and C. gloeosporioides f. sp. aeschynomene revealed no base substitutions at codons 198, 199, 200, and 240; however, sequence comparisons between C. truncatum isolates and C. gloeosporioides f. sp. aeschynomene revealed two codon changes located outside of the identified codon 198 or 200 associated with the benzimidazole-resistant phenotype of C. gloeosporioides isolates.