Inhibitory activity of Bacillus velezensis 33RB and Aspergillus niger 46SF was evaluated against numerous phytopathogens. The bacterial strain displayed the highest inhibitory activity toward Colletotrichum gloeosporioides BJ02 and Fusarium oxysporum 20RF (61.2 and 49.4%, respectively). Also, the maximum inhibitory activity of A. niger 46SF was exhibited (75.51 and 70.83%) against C. gloeosporioides BJ02 and F. oxysporum 20RF, respectively. The minimum volume (6.25 ml) of sterilized cultural filtrate of bacterial and fungal strains significantly inhibited the growth of C. gloeosporioides BJ02 by 73.3 and 83.3%, respectively, and F. oxysporum 20RF reached 40.4 and 78.8%, respectively. B. velezensis 33RB and A. niger 46SF displayed the highest inhibition toward C. gloeosporioides BJ02 and F. oxysporum 20RF at neutral pH and pH 5, respectively. Moreover, the highest inhibitory activity of B. velezensis 33RB and A. niger 46SF was achieved at 37 °C and 28 °C, respectively. Pathogenicity tests on sterilized detached leaves indicated that these isolates could potentially affect anthracnose and fusarium wilt diseases. Several secondary bioactive metabolites that assured the biocontrol efficacy of tested microbes were detected by liquid chromatography–mass spectrometry (LC–MS). The most detectable compounds included organic acids such as fumaric, DL-malic, citric, isobutyric, and glutamic acids. Also, numerous fatty acids such as lauric, linoleic, oleic, stearic, and myristic acids with diverse biological functions, including antimicrobial properties, were determined.