Lipid-dependent Malassezia species, Candida spp. and Rhodotorula mucilaginosa have been associated with bovine parasitic otitis. This paper evaluated the susceptibility of 63 yeast isolates from cattle with otitis to a povidone iodine aqueous solution (1% and 0.5% v/v) and to an alcohol-ether solution (1:1 v/v). The effectiveness of these antiseptics was assessed using the European suspension test. Products achieving equal to or greater than 5-log reduction in numbers of the challenge organism after 5 min contact are considered to have as acceptable microbicidal effect (ME). The two antiseptic solutions achieved ME greater than 5, when tested at 1 and 5 min contact time, against the majority of yeast strains. The exceptions were alcohol and ether solution against two Candida tropicalis strains. Urea broth macrodilution method was used to determine the minimum inhibitory concentration (MIC), defined as the lowest concentration that resulted in a visually negative urease test or, in the case of Candida spp., turbidity inhibition when compared with that produced by the growth control. Analysis of the results for all 63 isolates showed Malassezia sympodialis and Rhodotorula mucilaginosa to be more susceptible to povidone iodine and Malassezia furfur strains to be less susceptible. Malassezia sympodialis was significantly more susceptible to alcohol-ether solution than other species. This study showed the in vitro efficacy of alcohol-ether solution and povidone iodine and proposes the need for clinical evaluation of the topical treatment and control of bovine otitis with these antiseptics and their effects on the ear microbiota and the ear canal.