To investigate the biological activity of a novel 24‐membered macrolide compound, JBIR‐19, isolated from the culture broth of the entomopathogenic fungus Metarhizium sp. fE61, morphological changes in yeast cells were examined using the automated image‐processing program CalMorph. Principal components analysis was used to elucidate dynamic changes in the phenotypes, revealing two independent effects of JBIR‐19 in yeast cells: bud elongation and increased size of the actin region. Using a fitness assay, we identified the genes required for robust growth in the presence of JBIR‐19. Among these were CCW12,YLR111W, and DHH1, which are also involved in abnormal bud morphology. Based on these results and others, we predict intracellular targets of JBIR‐19 and its functional interactions.