Phenotype-guided natural products discovery is emerging as a useful new discovery tool that addresses challenges in early, unbiased natural product biological annotation. These high-content approaches yield screening results that report directly on the impact of test compounds on cellular processes in target organisms and can be used to predict the modes of action of bioactive constituents from primary screening data. In this study we explored the use of our recently implemented cytological profiling platform for the isolation of compounds with a specific, predefined mode of action, namely, induction of mitotic arrest. Screening of a microbially derived extract library revealed six extracts whose cytological profiles clustered closely with those of known antimitotic agents from the pure compound training set. Subsequent examination of one of these extracts revealed the presence of two separate bioactive constituents, each of which possessed a unique cytological profile. The first, diketopiperazine XR334 (3), recapitulated the observed antimitotic phenotype of the original extract, demonstrating that cytological profiling can be used for the targeted isolation of compounds with specific modes of action. The second, nocapyrone L (6), possessed a cytological profile that clustered with known calcium channel modulators, in line with previous published activities for this compound class, indicating that cytological profiling is a flexible and powerful platform for the de novo characterization of compound modes of action.