Imidazolium salts reveal interesting biological properties, especially regarding antitumor and antimicrobial activities. Two series of imidazolium salts based on steroids were obtained in an efficient and convenient synthesis. They were biologically tested to evaluate their antibacterial and antifungal properties. The activities of new salts, especially in relation to Gram-positive bacterial strains are comparable to the activities of known antibiotics. The most promising activity was that against C. albicans, which exceeded the antifungal activity of commonly used drugs. Some of the new salts exhibited improved antifungal activities against phytopathogenic fungi: B. cinerea and C. beticola. Our research showed that new compounds could be potentially useful as antifungal antibiotics or inhibiting agents against pathogenic fungi.