Thaumatin‐like proteins (TLPs) are part of the pathogenesis‐related (PR) proteins that are important in host plant defense. TLPs have multiple defense functions against pathogens (bacteria, viruses, and fungi) and are involved in other biological processes of several living organisms. However, the biological role of TLPs must be elucidated for other species, such as Phaseolus lunatus, popularly known as lima bean. In this study, we propose to investigate the biological activity of TLPs in P. lunatus, which can help develop a detailed functional characterization of the TLP gene in other crop cultures. As a result, a total of 31 PlTPLs genes and 16 highly conserved cysteine residues for each family member were identified in the lima bean genome. Regarding phylogenetic analysis, 128 TLPs from different plant species, including P. lunatus, were used, and the phenetic tree revealed that the TLPs could be divided into six groups. The leading group was Group 4, containing 34 TLPs; the smallest was Group 7, containing only a single TLP. The analysis of antimicrobial and antiviral activity revealed that 27 PlTLPs have antimicrobial activity and 7 PlTLPs have potential antiviral activity. For chromosomal distribution of the PlTLPs, 8 of 11 chromosomes presented at least one PlTLs on one of the chromosomes. Finally, molecular modeling was carried out between two TLPs (PlTLP5 and PlTLP14), which presented antimicrobial and antiviral activity and did not have allergenic activity. The results showed that the two candidates presented ≥90% of their residues in the favorable regions and ∼10% in the permitted regions, demonstrating that the two PlTLPs are considered promising targets for biotechnological applications, especially those related to the genetic improvement of agricultural crops and the development of antimicrobial agents.