Background/Objectives: pentane polyene antibiotic Roseofungin produced by actinomycetes possessing wide range of antimicrobial activity. Methods: The structure of novel polyene antibiotic Roseofungin was confirmed through FTIR, H¹ nuclear magnetic resonance, and high-performance liquid chromatography analysis with a mass detector. The substance pharmaco-technological parameters were evaluated. Additionally, the in silico modelling of various physicochemical parameters and biological activities was performed using validated open-access software tools such as ProTox3, SwissADME, and ADMET SAR1. The evaluation of its toxicological profile was also investigated in vivo. Results: The Roseofungin exhibits potential risks in certain categories, including immunotoxicity, respiratory toxicity, and nephrotoxicity, as predicted in silico. However, Roseofungin shows a relatively safe profile with regard to hepatotoxicity, neurotoxicity, and mutagenicity, along with lower risks of carcinogenicity and cytotoxicity in silico. The analysis of body weight dynamics after Roseofungin exposure in mice revealed no statistically significant differences among the experimental groups. Similarly, in the absolute or relative weights of internal organs across the experimental groups after Roseofungin treatment, no significant differences were observed in vivo. Roseofungin appears as a light-yellow hygroscopic powder with a specific odour, possessing the ability to settle and classified as a light powder. The particles are lamellar crystals ranging in size from 3 μm to 6 μm, and the molecules generate electrostatic tension between themselves. The pharmaco-technological parameters of Roseofungin were comprehensively studied. Conclusions: The experimental data obtained provide a foundation for further pharmaceutical development of new drugs containing the original Roseofungin.