Gray mold is a common disease of many crops worldwide. Tetramycin is a broad-spectrum biopesticide that may control gray mold disease. In the present study, in vitro biological activity of tetramycin was measured in three development stages of Botrytis cinerea, and the baseline sensitivity of B. cinerea to tetramycin was characterized with a set of 165 isolates that were obtained from different geographical regions in Shandong Province, China. Tetramycin’s protective and curative activity against the fungal pathogen was determined on strawberry fruit in laboratory trials, and the efficacy of tetramycin was also determined in field conditions. Spore germination was found to be the most sensitive growth stage that was inhibited by tetramycin, whereas the mycelial growth was the least sensitive growth stage. The baseline sensitivities showed that the frequency distributions of tetramycin were unimodal curves, with mean EC50 values of 0.35 ± 0.20 and 0.020 ± 0.009 μg mL−1 for the inhibition of mycelial growth and spore germination, respectively. Tetramycin had no cross-resistance with other fungicides tested, including carbendazim, iprodione, diethofencarb, pyrimethanil, pyrisoxazole and boscalid. In field trials performed in Tai’an city, Shandong province in 2014 and 2015, tetramycin was used at a concentration of 60 g a.i. ha−1, provided gray mold with an efficacy ranging from 70.73 % to 78.44 %, with no significant difference with other fungicide treatments. These results showed that tetramycin may serve as an alternative fungicide for the control of gray mold in strawberry fruits and cucumber.