Chinese kale black spot disease is caused by the fungus Alternaria brassicicola (Schw.), which is one of the most significant destructive pathogens that attacks vegetable crops, especially Chinese kale. Currently, the pathogen management is achieved by using synthetic fungicides, but these are harmful to human health and tend to be expensive. Consequently, there is an urgent need to find alternative management options. The objective of this study was to evaluate the antagonistic activity of Talaromyces flavus (Klöcker) Stolk and Samson Bodhi001, Talaromyces trachyspermus (Shear) Stolk and Samson Bodhi002, Talaromyces flavus (Klöcker) Stolk and Samson Bodhi003, Neosartorya fischeri (Wehmer) Malloch and Cain, Bodhi004, Eupenicillium sp., and Gongronella butleri (Lendn.) Peyronel and Dal Vesco in in vitro tests for the control of A. brassicicola causing Chinese kale black spot disease under greenhouse conditions. The in vitro tests showed that among the tested pathogens, T. flavus Bodhi001 inhibited the mycelial growth of A. brassicicola by 65% in a dual culture method and formed an inhibition zone 0.8–0.9 cm wide. Under greenhouse conditions, spore suspensions of 10⁶ spores mL⁻¹ of Eupenicillium sp., T. flavus Bodhi001, T. trachyspermus Bodhi002, N. fischeri Bodhi004, G. butleri, and T. flavus Bodhi003 effectively reduced and suppressed the incidence of black spots caused by A. brassicicola at 30 days after transplanting (DAT). However, the greatest suppression of the development of black spots in terms of disease incidence was 32.56% and occurred when plants were treated with the spore suspension of T. flavus Bodhi001 once at 30 DAT and again at 40 DAT compared to a water control. The results of this study indicated that T. flavus Bodhi001 could provide protection for Chinese kale, and is a promising biological control agent against A. brassicicola.