Biological activities of bombolitins from Bombus ardens, B. consobrinus, B. terrestris and B. ussurensis (bombolitins A, C, T and U, respectively) were examined using hemolytic, anti-microbial, anti-fungal and anti-tumor activity assays. Among the four bombolitins tested, bombolitin T showed the highest hemolytic and anti-tumor activities. All bombolitins exhibited strong anti-microbial and anti-fungal activities, and bombolitin A specifically possessed the highest anti-microbial activity against the Gram-negative bacteria Escherichia coli. Circular dichroism spectrometry analysis revealed that all four bombolitins had over 61.7% and 45.5% of α-helicity in 30 mM sodium dodecyl sulfate and 50% trifluoroethanol buffers, respectively, which form lipid-membrane-mimicking environments. Bombolitin T showed the lowest IC50 values of 8.5 μM and 8.8 μM against SK-OV-3 and NIH-OVCAR-3 cell lines, respectively, after 72 h of treatment, but its relative hemolytic activity at a concentration of 200 μM was 2.3-fold higher than that of 0.1% Triton X-100. To design selective anti-tumor peptides with reduced hemolytic activity, bombolitin T was chosen as a model to be modified by amino acid substitution. The lowest hemolytic activity was observed with the addition of a hydrophobic amino acid (Ile14) to the C-terminal end as well as the substitution of Ser10 and Leu12 with a hydrophobic amino acid (Leu) and positively charged amino acid (Lys), respectively. This finding implies that the balanced substitution of hydrophobic and positively charged amino acids could affect the cytotoxicity. This study provides new information on the properties of anti-tumor peptides in the venoms of bumblebees and the basic approaches for peptide design tools for the reduction of cytotoxicity.