ABSTRACTInfections withScedosporiumspp. andLomentospora prolificanshave become a serious threat in clinical settings. The high mortality rates associated with these infections can be correlated with their multidrug resistance. The development of alternative treatment strategies has become crucial. Here, we investigate thein vitroandin vivoactivity of luliconazole (LLCZ) againstScedosporium apiospermum(including its teleomorphPseudallescheria boydii) andLomentospora prolificans. The LLCZ MICs were determined for a total of 37 isolates (31L. prolificansisolates, 6Scedosporium apiospermum/P. boydiistrains) according to EUCAST. Furthermore, the LLCZ antifungal activity was testedin vitro, using an XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt] growth kinetics assay and biofilm assays (crystal violet and XTT assay). In addition, aGalleria mellonellainfection model was used forin vivotreatment assays. The MIC90of LLCZ was determined to be 0.25 mg/L for all tested pathogens. Growth was inhibited within 6 to 48 h of the start of incubation. LLCZ inhibited biofilm formation in both preadhesion stages and late-stage adhesion.In vivo, a single dose of LLCZ increased the survival rate of the larvae by 40% and 20% forL. prolificansandScedosporiumspp., respectively. This is the first study demonstrating LLCZ activity againstLomentospora prolificans in vitroandin vivoand the first study showing the antibiofilm effect of LLCZ inScedosporiumspp.IMPORTANCELomentospora prolificansandS. apiospermum/P. boydiiare opportunistic, multidrug-resistant pathogens causing invasive infections in immunosuppressed patients and sometimes in healthy persons.Lomentospora prolificansis panresistant against the currently available antifungals, and both species are associated with high mortality rates. Thus, the discovery of novel antifungal drugs exhibiting an effect against these resistant fungi is crucial. Our study shows the effect of luliconazole (LLCZ) againstL. prolificansandScedosporiumspp.in vitro, as well as in anin vivoinfection model. These data reveal the previously unknown inhibitory effect of LLCZ againstL. prolificansand its antibiofilm effect inScedosporiumspp. It represents an extension of the literature regarding azole-resistant fungi and could potentially lead to the development of future treatment strategies against these opportunistic fungal pathogens.