A series of novel hybrids β-carboline-4-thiazolidinones were synthesized and evaluated for their in vitro antitumor activity against human cancer cell lines and for antiviral activity towards Herpes simplex virus type-1 (HSV-1). From the N'-(2-ylidene-4-thiazolidinone)-β-carboline-3-carbohydrazide series (9-11), compounds 9c and 11d were the most active, showing growth inhibition 50% (GI50) values less than 5 μM for all cell lines tested. Compound 9c, bearing the 4-dimethylaminophenyl group at C-1 of β-carboline was selected for further investigation concerning cell death and cell cycle profile, focusing on the human renal adenocarcinoma cell line 786-0. Treatments with 25 μM of compound 9c induced cell death after 15 h of treatment, characterized by phosphatidylserine exposure and loss of membrane integrity. Moreover, treatment with 12.5 μM promoted a sub-G1 arrest, which indicates cell death. Derivatives of the N-(2-substituted-aryl-4-thiazolidinone)-β-carboline-3-carboxamide series (18-23) showed a potent activity and high selectivity for glioma (U251) and ovarian (OVCAR-3) cancer cell lines. Also, some β-carboline-4-thiazolidinone hybrids showed potent antiviral activity against Herpes simplex virus type-1. The N-(2-substituted-aryl-4-thiazolidinone)-carboxamide moiety in 18, 19 and 22 confer a potent anti-HSV-1 activity for these derivatives, which presented EC50 values of 0.80, 2.15 and 2.02 μM, respectively. The assay results showed that the nature of 4-thiazolidinone moiety and of the substituent attached at the 3- and 1- position of β-carboline nucleus influenced the antitumor and antiviral activities.