The identification of heterocyclic small molecules that cover unexplored chemical space is of great importance for the development of new small-molecule therapeutics. In this study, we synthesized a series of 5-amino-1,3,4-thiadiazoles appended isatins (UZ-1-20) that exhibited polypharmacological properties, as evaluated in a cell-painting assay assessing induced cellular morphological changes. A high hit rate ranging from 55% to 80% was observed for the tested compounds at varied concentrations. The most active compounds showed significant activity in inducing cellular morphological changes with a measured induction value of more than 30% and shared a high biological profiling similarity with an antifungal agent itraconazole and a chemokine receptor inhibitor. The synthesized compounds exhibited moderate to good antiproliferative activity against tested cancer cell lines in the MTT assay. Molecular docking studies were performed to theoretically probe and compare the binding modes between the most active UZ compounds and ITZ or BI-6901, respectively. Additionally, ADMET analysis indicated favorable pharmacokinetic parameters including good oral bioavailability, balanced hydrophilicity, and minimal toxicity. Overall, the findings in this study highlight the potential of developing the aminothiadiazole appended isatins as bioactive agents.