Our study aimed at selecting new biological control agent (BCA) strains for their inhibitory activities against Pythiaceae species causing apple dieback as new sources to control this important disease of Tunisian apple orchards. We focused on antibiosis as a mode of action of selected isolates obtained from our screening. The fungal isolates Trichoderma simmonsii A2, T. asperellum A3, and Aspergillus westerdijkiae A7 showed the strongest inhibition profiles against the tested Pythiaceae isolates with respect to inhibition rates and activity spectra in in vitro inhibition assays using the isolates or their culture filtrates. In particular, A. westerdijkiae A7 culture filtrate inhibited growth of Pythiaceae isolates by up to 100%. T. simmonsii A2, A. westerdijkiae A7, and one bacterial isolate ( Bacillus sp. strain B2) were tested for their protective effect in planta against dieback disease on apple rootstocks alone or in combination as preventive and curative applications. The results revealed that T. simmonsii A2 exhibited the strongest preventive activity against Pythium undulatum BME and Phytophthora inundata BGA1 in solo application. The combination of different BCAs had contrasting effects on disease control, indicating additive or synergistic effects between two BCAs but negative interactions in the triple combination. Our study also revealed that the three tested BCAs had plant growth-promoting activities on root and shoot parameters, alone or in combination. Altogether, our study provides promising leads for new BCAs. The BCA combination is of particular interest for further studies aiming at the isolation of new molecules with anti-oomycete and/or plant growth-promoting activity. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .