Abstract Objectives We sought to evaluate the comparative activity of rezafungin compared with caspofungin and other antifungal classes against biofilms from a large clinical panel of Candida strains (n = 167). Methods Biofilm killing and inhibition were assessed using standard XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt] metabolic assessment. Biofilm time–kill kinetics were also evaluated using metabolic and viable cell counts. Microscopy was performed to visually assess biofilm inhibition. Results Rezafungin was shown to outperform caspofungin and other antifungals against C. albicans, C. parapsilosis, C. tropicalis and Nakaseomyces glabratus (previously called C. glabrata) strains with a heterogeneous biofilm phenotype. Assessment of high biofilm-forming strains at 0.03 mg/L concentrations showed that rezafungin killed biofilms to an equal or greater extent than caspofungin. Time–kill studies showed a rapid reduction in metabolism and viable cfus by both rezafungin and caspofungin, but with little difference between both compounds. Evaluation of biofilm inhibition characteristics of both compounds showed that rezafungin was marginally more effective than caspofungin, which was corroborated by microscopical analyses. Conclusions Together, these data show that rezafungin is non-inferior to caspofungin in terms of anti-biofilm activity and displays characteristics that suggest it can control biofilms more effectively than caspofungin. Further evaluation is required to establish whether these in vitro effects translate clinically, but the data indicate an opportunity for rezafungin to be used for the clinical management of biofilm-related diseases.