Guanidines are fascinating small nitrogen-rich organic compounds, which have been frequently associated with a wide range of biological activities. This is mainly due to their interesting chemical features. For these reasons, for the past decades, researchers have been synthesizing and evaluating guanidine derivatives. In fact, there are currently on the market several guanidine-bearing drugs. Given the broad panoply of pharmacological activities displayed by guanidine compounds, in this review, we chose to focus on antitumor, antibacterial, antiviral, antifungal, and antiprotozoal activities presented by several natural and synthetic guanidine derivatives, which are undergoing preclinical and clinical studies from January 2010 to January 2023. Moreover, we also present guanidine-containing drugs currently in the market for the treatment of cancer and several infectious diseases. In the preclinical and clinical setting, most of the synthesized and natural guanidine derivatives are being evaluated as antitumor and antibacterial agents. Even though DNA is the most known target of this type of compounds, their cytotoxicity also involves several other different mechanisms, such as interference with bacterial cell membranes, reactive oxygen species (ROS) formation, mitochondrial-mediated apoptosis, mediated-Rac1 inhibition, among others. As for the compounds already used as pharmacological drugs, their main application is in the treatment of different types of cancer, such as breast, lung, prostate, and leukemia. Guanidine-containing drugs are also being used for the treatment of bacterial, antiprotozoal, antiviral infections and, recently, have been proposed for the treatment of COVID-19. To conclude, the guanidine group is a privileged scaffold in drug design. Its remarkable cytotoxic activities, especially in the field of oncology, still make it suitable for a deeper investigation to afford more efficient and target-specific drugs.