► A novel class of venom peptides (referred to as HsAp, HsAp2, HsAp3 and HsAp4) were identified from Heterometrus spinifer. ► The four peptides are highly basic, cationic and weakly amphipathic. ► HsAp processes antimicrobial activities against both Gram-positive and Gram-negative bacteria, as well as a fungus. ► The genes encoding the four peptides are intronless. The venom peptides from the scorpion Heterometrus spinifer have been poorly characterized so far. Here, we identified a novel class of antimicrobial peptides from the venom gland of H. spinifer, which were referred to as HsAp, HsAp2, HsAp3 and HsAp4, respectively. Each of the four peptides consists of 29 amino acid residues, and is cationic and weakly amphipathic. They display no significant homology to any other known peptides, and thus represent a new family of venom peptides from scorpions. Antimicrobial assay showed that HsAp is able to inhibit the growth of both Gram-negative and Gram-positive bacteria with the MIC values of 11.8–51.2μM. HsAp is also able to inhibit the growth of the tested fungus. Genomic analysis indicated that the genes of all the four peptides are intronless. Our studies expand the families of antimicrobial peptides from scorpions.