Actinomyces, particularly Streptomyces, have a major impact on protecting plant roots by inhibiting the growth of fungal species based on their capacity to release antifungal antibiotics. Herein, FT-IR spectra for Streptomyces rochei extract revealed the varied patterns of functional group absorption of the bioactive compounds (BACs). Furthermore, using LC-ESI-MS/MS analysis, 21 novel diverse secondary metabolite compounds from S. rochei extract were found: nine flavonoids, one flavonoid derivative, one phytohormone, eight phenolic acids, one benzoic acid esters and one triterpenoid. Likewise, a DPPH radical scavenging capacity experiment was conducted to assess the antioxidant properties of four different concentrations (50, 100, 150 and 200 µg/ml) of S. rochei extract. In addition, BACs from S. rochei exhibited significant antibacterial activity against dangerous human pathogens Bacillus cereus, Staphylococcus aureus and gram-negative bacteria Salmonella typhimurium. Also, antimycotic activity against Candida albicans was measured. S. rochei extract showed a moderate level of cytotoxicity against lung cancer cell lines (A-549) with low cytotoxic effects on lung normal cell lines (WI-38). The biological activities of streptomyces extract might be attributed to the presence of a variety of metabolites, and hence, S. rochei can be used as an excellent source of bioactive metabolites.