Novel betulinic, ursolic, and oleanolic acid derivatives, containing a guanidine moiety have been designed and synthesized in an attempt to develop potent antitumor, antibacterial and antifungal agents. Triterpenoic acids were converted into C-28-aminotriterpenoids in which polyamine moieties were bound with C-28 carboxylic group through an amide or ester bonds. These compounds served as precursors for the synthesis of novel guanidine-functionalized triterpenoic acids derivatives. The cytotoxicity was tested on five human tumor cell lines (Jurkat, K562, U937, HEK, and Hela) and compared with the tests on normal human fibroblasts. The antitumor activities of the most tested guanidine derivatives was lower than that of corresponding amines, but triterpenoids with the guanidine group were less toxic to human fibroblasts. The identified lead molecules with the highest antitumor characteristics were selected for extensive biological testing according to flow cytometry data, which showed that the antitumor activity of these compounds is caused by apoptotic processes and induction of cell cycle arrest in the S-phase. Most of the tested guanidine derivatives showed a good antibacterial effect against Gram-positive bacteria Staphylococcus aureus (MICs values 0.5-4.0 mg/mL) and expressed significant antifungal activity against Candida albicans (4.0 mg/mL) and Cryptococcus neoformans (0.25-4.0 mg/mL), higher than the standard fluconazole (8.0 mg/mL).