Clinical infectious diseases are a major public health concerns worldwide, particularly in developing countries, where access to health care is extremely limited. In a context of increased development of antibiotic-resistant microorganisms, extensive research efforts, to explore novel antimicrobial from newly isolated organisms, are in need. Therefore, our study was carried out to isolate actinobacteria, for the first time, from two different soils collected from Cameroon. Fifty-eight out of 81 isolates of actinobacteria showed antimicrobial activity against human pathogens based on agar disc overlay bioassay. Meanwhile, the excreted metabolites of these actinobacteria were also tested for antimicrobial activity using agar disc diffusion test. Nine Gram-positive and negative human bacterial pathogens were used. One fungal pathogen was also tested. Our results showed that the most active actinobacterial isolates are belonging to the genera: Microtetraspora sp. (CSU3), Nocardiopsis sp. (CSU5), Streptomyces sp. (PSP3, PSU28 and PSU30), and Micromonospora sp. (CSD50). Streptomyces sp. and Actinomadura sp. isolates were the most dominant genera producing potent antimicrobial secondary metabolites, indicating that Streptomycetes-phenotype isolates are providing high quality metabolites for drug discovery purposes. Natural occurring biochemical compounds excreted in liquid media were also tested and proved the presence of alkaloids and tannins compounds.