Multi-drug resistance of pathogenic microorganisms is a serious threat to human health. In particular, Candida albicans, the most common opportunistic clinical fungal pathogen is one of the major causes of systemic infections, resulting in an estimated 30% of severe fungal infections, with mortality rate reaching nearly 40%. Widespread and repeated use of current drugs, particularly azoles, have contributed to the rapid occurrence of antifungal drug resistance, and most screening approaches for new drugs which target essential genes and fungal pathogens are likely to generate resistance over time. Given the high mortality rate resulting from fungal infections in immunocompromised patients and the limited number of highly effective, yet safe treatment agents, the development of new antifungal therapeutics is critical. A strategy limiting the pressure on drug targets would increase the lifespan of antifungal agents and reduce the frequency of treatment failures. For instance, using synergistic drugs aimed at more than one target and slowing down the emergence of drug-resistant pathogens will be one of the key approaches to antifungal therapy. Looking for natural products with potentially new Mode of Action from untapped sources provides an essential strategy. Endophytic microbes associated with traditional Chinese medicines provide a variety of active compounds for therapeutics. A high-throughput synergy screening (HTSS) method has been developed in our lab for identifying synergistic microbial natural products in combination with a low dosage of ketoconazole against fungal pathogens in vitro. Further screening on a sub-library of microbial natural products has been done, contained 900 crude extracts from the fermentation broth of endophytic bacteria 300 strains on three different media (AM2, NM2 and MPG). HTSS on these extracts gave a 1.2% hit rate and generated 90% of the maximum activity in combination with 1/4 MIC of KTC. In addition, the microbial extract alone had little to no effect. Among these hits, along with the chemo-type analysis, an extract derived from the fermentation broth of endophytic actinomycete Streptomyces sp. (CNS-42) associated with traditional Chinese medicine Alisma orientale was highlighted with MIC values of 6.25 and 3.125 μg ml − 1 against C. albicans (SC 5314) and C. albicans (SC 5314) synergistic model, respectively. Bioassay-directed fractionation of a large-scale (42 L) culture of CNS-42 yielded four known indolecarbazoles (1–4). 1 and 2 showed potent synergistic antifungal activities with MIC values of 6.25 and 0.78 μg ml − 1, respectively, when combined with the addition of 25% of the MIC of ketoconazole in the fungal suspension, but weak or no antifungal activity (MIC⩾100 μg ml − 1) when tested singly. This paper describes the finding of new activity of indolecarbazoles for further pharmaceutical application study.