In the context of the sixth mass extinction of animals, programs for the conservation and breeding of amphibians – one of the most vulnerable groups of vertebrates – have become particularly relevant. A key challenge in the application of assisted reproductive technologies in anurans is the asynchronous maturation of gametes between males and females, which could be mitigated by developing effective sperm storage protocols. This review summarizes and analyzes research findings on the effects of antibiotics on the preservation of anuran spermatozoa during hypothermic storage (0–5°C). It has been shown that initial failures in the use of antibiotics for storing spermic urine were due to high concentrations toxic to spermatozoa (1–4 mg/ml). The application of broad-spectrum antibiotics, such as gentamicin, streptomycin, and enrofloxacin, at lower doses (below 1 mg/ml) was found to suppress bacterial growth and significantly increase the viability of spermatozoa – up to 1.5 months for Rana temporaria and 2 weeks for Bufo bufo, while maintaining their motility and fertilizing capacity. Gentamicin at concentrations within 0.1–0.4 mg/ml proved to be the most effective among the studied drugs. Future perspectives include optimizing storage protocols to account for species-specific characteristics, identifying new non-toxic antimicrobial agents, and investigating combined antibiotic formulations.