Two new β-class milbemycins from Streptomyces bingchenggensis, named milbemycin β₁₃ and β₁₄, have been isolated and characterized. The producing organism has been deposited at the China General Microbiology Culture Collection Center (Accession No: CGMCC1734). On the basis of detailed spectroscopic analysis and comparison with reported data, their structures were determined to be the hydroxyl derivatives at C-27 of milbemycin β₃ and 25-ethylmilbemycin β₃, respectively. Milbemycins β₁₃ and β₁₄ possess potent acaricidal and nematocidal activity. The discovery of these two compounds plays an important role in understanding and perfecting the proposed pathways of milbemycins.