Streptomyces thermoviolaceus SRC3, a newly isolated actinobacterial strain from Algerian river sediments, exhibited a broad activity against various bacterial and yeast human pathogens (Salmonella Typhi ATCC 14028, Vibrio cholerae ATCC 14035, MRSA ATCC 43300 and Candida albicans ATCC 10231). The strain SRC3 was selected from thirty nine actinobacterial isolates and identified as S. thermoviolaceus based on morphology, cultural properties, physiological analyses and 16S rRNA gene sequencing. Culture parameters for the antibiotic production were optimized by sequential statistical strategy including Plackett-Burman design (PBD) and Response Surface Methodology (RSM). In PBD experiments, KCl, K2HPO4, MgSO4 center dot 7H(2)O, pH value and incubation time emerged as the most significant in affecting the output of antimicrobial activities.These factors were further optimized using Central Composite Design (CCD). The best achieved conditions were: KCl (0.01%), K2HPO4 (0.1%), MgSO4 center dot 7H(2)O (0.02%) and 9 days incubation for anti-S. Typhi compounds, KCl (0.051%), MgSO4 center dot 7H(2)O (0.05%) and 5 days incubation for C. albicans inhibitors. The metabolite responsible for the bioactivities was purified, structurally characterized (by NMR, MS, UV and IR analyses) and identified as streptazolin, recently reported as a promising antibiotic adjuvant.