Anthrax is a worldwide distributing zoonotic disease, caused by Bacillus anthracis, which occurs sporadically in Indonesia, particularly in the provinces of East Java, Central Java, and Yogyakarta, which are the working areas of the Disease Investigation Center (DIC) Wates. Penicillin has been the primary antimicrobial treatment recommended for anthrax since there has never been a report of resistance to this antibiotic in Indonesia. The objective of this research was to assess the sensitivity of B. anthracis isolates from Central Java, East Java, and Yogyakarta to penicillin and tetracycline. Sixteen B. anthracis isolates from DIC Wates collected between 1990-2021 recovered from environmental samples were used in this study. All isolates were identified by phenotype, then tested for sensitivity to penicillin and tetracycline by agar diffusion (Kirby-Bauer) and broth dilution method. The data obtained were compared with the standard and analyzed descriptively. The results showed that all isolates were B. anthracis. One of 16 isolates (6,25%) consistently showed resistance to penicillin, but was sensitive to tetracycline, while 15 isolates (93,75%) showed sensitive to both antibiotics. A penicillin-resistant isolate was soil sample from anthrax endemic area. In conclusion, there was B. anthracis isolate that was found resistance to penicillin. Therefore, tetracycline can be used as an alternative for anthrax treatment.