Abstract: Abstract: Gray mold, caused by Botrytis cinerea Persoon, is an important preharvest and postharvest disease on fresh tomato. Using chemical fungicide is the most common strategy to effectively minimize the severity of B. cinerea, but is not considered to be a long term solution because of the potential health and environmental risk. Moreover, fungicide-resistant strains have emerged by using chemical fungicide. It is necessary to search for alternative methods. Macrolides are an effective and safe class of antibiotics, mainly produced by actinomycetes and fungi. Their structures and biological activities are highly diverse. To date, more than 200 kinds of known macrolide antibiotics (e.g., rapamycin, nystatins, filipins and amphotericin B) have been isolated and characterized, most of which are produced by the genus Streptomyces. Novonestmycin A is a new 32-membered macrolide compound produced by Streptomyces phytohabitans HBERC-20821, with the molecular weight of 1 228.66. In previous study, it showed inhibition activity on some plant pathogenic fungi in vitro. In order to explore the application of Novonestmycin A on controlling gray mold of tomato, the bioassay was conducted in vitro and in fruit. It strongly inhibited mycelial growth of Botrytis cinerea in vitro. The concentration of fungicide was set to 2.5, 1.25, 0.625, 0.312 5 and 0.156 25 μg/mL, the median inhibitory concentration was calculated as 0.631 g/mL, and the toxic regression equation was established with the correlation coefficient of 0.999 7. The median inhibitory concentration and the correlation coefficient of toxic regression equation for carbendazim were 0.157 μg/mL and 0.996 1 respectively. When treated with Novonestmycin A with the concentration of 1.25 μg/mL, it was observed that hyphae morphology became irregular with optical microscope. And it also had excellent control effect on tomato gray mold in fruit. The tomato fruit was pierced and soaked with Novonestmycin A with the concentration of 50 and 100 μg/mL, respectively. When the tomato was dried in the air, it was inoculated with the spore suspension of Botrytis cinerea of 10 μL, and the fruit was cultured with moisture preservation at 25 ℃ for 6 d. It showed that the control effect was 68.29% and 100% respectively at the concentration of 50 and 100 μg/mL and the control effect was equivalent to carbendazim. Novonestmycin A had no harm to epidermis of tomato under test concentration. It also has a broad fungicide spectrum. The inhibition rate on Sclerotinia sclerotiorum, which belongs to ascomycetes, was 86.11%. The inhibition rate on Corynespora cassiicola, Alternaria solani, Colletotrichum gloeosporioides and Pestalotiopsis guepinii, which belong to deuteromycetes, were 91.11%, 82.78%, 77.22% and 86.46% respectively. The inhibition rate on Rhizoctonia solani, which belongs to basidiomycetes, was 85.56%. But it had no effect on Phytophthora capsici and Ralstonia solanacearum, which belong to oomycetes and bacteria. Novonestmycin A has excellent control effect on grey mold of tomato fruit and has no injury on epidermis of fruit. It also has inhibition effect on several plant pathogenic fungi and hence has broad application prospects. Thus it has potential as a new fungicide in the controlling of tomato gray mold.