Multispecies biofilms, in which both fungus and bacteria species can be present, play a significant role in persistent infections, and new therapeutic options are needed against them. In this study, the activities of ceragenins and antimicrobial peptides (AMPs) (magainin, cecropin A, LL-37) were investigated against multispecies biofilms formed by Candida albicans and four clinically important Gram-negative bacteria, including Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae. Our results show that CSA-13 and CSA-90 were the most effective agents against both mono and multispecies biofilms (P < 0.05). CSA-131 and CSA-192 showed the least antimicrobial activity against mono and fungal-bacterial multispecies biofilms. Inhibition of multispecies biofilms with CSA-13 and CSA-90 was also confirmed through fluorescence microscopy images. When AMPs evaluated alone, they proved ineffective against both C. albicans and Gram-negative bacteria at the concentrations tested. In these studies, ceragenins were much more effective than AMPs against multi or monospecies biofilms, especially those containing C. albicans.