Indeed, Actinomycetes and Penicillium are microorganisms that live in different ecosystems. Besides, they play important roles in various fields in respect such as agriculture, medicine and the environment. During this study, we were interested in the isolation of two strains namely: Actinomycetes and Penicillium sp. from coconuts and truffle respectively. In addition, the agar cylinder technique demonstrated the antibacterial activity of Penicillium sp. against four pathogenic bacterial strains (S. aureus, E. coli, P. aeruginosa and K. pneumoniae). In virtue of which, the results showed a variability of the diameters of the inhibition zones, thus the most important results were recorded against K. pneumoniae and S. aureus, which produced inhibition zones of 17.50±0.71 mm and 17.00±0.00 mm respectively. In order to struggle against pathogenic fungal strains, the ability of Actinomycete strain to suppress the mycelial growth of 03 pathogenic strains of Fusarium oxysporum f.sp. albedinis (EF1, EF2 and EF3) was examined. Hence, the result of the direct confrontation tests on the PDA medium between the Actinomycete strain and these pathogenic strains revealed a significant inhibition of the mycelial growth of EF3 (62,5%) and EF2 (52,70%). On the other hand, the lowest inhibition rate was recorded in EF1 (34.55%).