Updated project metadata. Acanthoscurria juruenicola is an Amazonian tarantula spider described for the first time a century ago. Specimens of both genders are similar in size and in most morphological aspects, but ecological behavior and their venom composition remained unknown to date. Here we present the peptidomics characterization of the spider venom by a combination of mass spectrometric analysis of both native and digested peptides, venom gland transcriptomics and bioinformatics. A total of 367 native features were observed in the venom peptidome. Seventeen cysteine-rich peptides were simultaneously observed in the transcriptome and in the mass spectrometric experiments, from which fourteen were completely sequenced in the mature forms. The mature peptides have 3-5 disulfide bonds and cover the 3.7-8.6 kDa mass range. Moreover, in vivo paralytic activities of the whole venom were observed in crickets. In silico analysis indicated that all mature peptides are potentially antimicrobial and two may be potential anticancer agents. The antimicrobial activity was experimentally confirmed for the peptide Ap1a against Micrococcus luteus, Pseudomonas aeruginosa and Candida albicans.