Loxoscelism is a recognized public health problem in Brazil, but the venom from Loxosceles similis, which is widespread in Brazil due to its adaptability to the urban environment, remains poorly characterized. Loxtox is a family of phospholipase D enzymes (PLDs), which are the major components of Loxosceles venom and are responsible for the clinical effects of loxoscelism. Loxtox toxins correspond to 15% of L. similis venom gland transcripts, but the Loxtox family of L. similis has yet to be fully described. In this study, we cloned and functionally characterized recLoxtox s1A and recLoxtox s11A. These recombinant toxins exhibited different in vitro activities depending on pH, and recLoxtox s1A had more intense effects on rabbit skin than did recLoxtox s11A in vivo. Both recombinant toxins were used in immunization protocols, and mapping of their epitopes revealed different immunological reactions for the produced immune serums. Additionally, polyclonal antibodies raised against recLoxtox s1A had greater capacity to significantly reduce the in vitro and in vivo effects of L. similis venom. In summary, we obtained and characterized two novel Loxtox isoforms from L. similis venom, which may be valuable biotechnological and immunological tools against loxoscelism.