Streptomyces parvulus AL036, isolated from Alpinia galanga root tissues, exhibited morphological, physiological, biochemical characteristics, and 16S rDNA sequence comparable to those of Streptomyces parvulus Tc022. Concordantly, strain AL036 displayed actinomycin D production capabilities similar to those observed in S. parvulus Tc022. (Taechowisan et al., 2006). Given the comparable morphological, physiological, biochemical, 16S rDNA sequence, and actinomycin D production profiles observed, Streptomyces parvulus AL036 and Tc022 may represent identical strains persisting within the Alpinia galanga root niche over a period of seventeen years (since the initial isolation in 2006). Furthermore, we identified a known antibiotic; actinomycin D produced by a Streptomyces parvulus AL036 isolated from the root tissues of Alpinia galanga. The compound was confirmed based on ESI-HRMS and NMR data as well as its bioactivity. Actinomycin D is one type of chromopeptide lactone antibiotics, which more than 30 native variants. All known natural actinomycins contain the same phenoxazone chromophore and differ only in the amino acid content of the peptide side chains (Mauger and Thomas, 1981). This antibiotic can be produced by different species of Streptomyces (Kurosawa et al., 2006) and Micromonospora (Wagman et al., 1976) as part of a mixture of several actinomycins (Korzybski et al., 1967; Praveen et al., 2008a). In this study, we report isolation, taxonomy of the producer organism, purification and characterisation of the anticancer actinomycin D by Streptomyces parvulus AL036. Actinomycin D exerts its antimicrobial activity by binding to DNA, thereby inhibiting RNA synthesis (Goldberg et al., 1962). Notably, it holds historical significance as the first clinically used antibiotic in 1954, and it continues to be a valuable tool in both cancer treatment and biochemical/molecular biological research (Koba and Konopa, 2005). The yield of actinomycin D with different species of Streptomyces varies substantially. Streptomyces parvulus, S. felleus and S. regensis have been shown to produce 152, 20 and 12 mg/mL of actinomycin D, respectively. Streptomyces parvullus produces actinomycin D almost exclusively (Williams and Katz, 1997; Sousa et al., 2002). However, most can produce only small quantities of actinomycin D. This study investigated the influence of carbon and nitrogen (C&N) sources on actinomycin D production using SC medium, which was identified as the most favorable based on initial experiments. Optimizing the concentrations of C&N nutrients and monosaccharide supplements is crucial for maximizing actinomycin D yield in both laboratory and industrial fermentation processes. Altering any of these parameters can significantly impact cell growth and production stability. In previous research by Sousa et al. (2002) who optimized the production medium for Streptomyces parvulus DAUFPE 3124, the initial composition consisted of 30 g/L soy milk, 20 g/L glucose, and 2 g/L CaCO3, resulting in a maximum actinomycin D concentration of 530 mg/L. Substitution of glucose with fructose at concentrations of 20, 30, and 40 g/L consistently yielded higher actinomycin D concentrations compared to the glucose-containing medium. Notably, the optimal concentration was determined to be 30 g/L fructose, leading to a maximum production of 635 mg/L (Sousa et al. (2002). Supplementation of SC medium with 0.5–1.0% glucose, galactose, or fructose conversely inhibited actinomycin D production but stimulated mycelial growth. Supporting our findings, Gallo and Katz (1972) reported similar observations of glucose-mediated repression of actinomycin D production. This reduction is attributed to the ~ 94% repression of phenoxazinone synthase, the enzyme responsible for catalyzing the synthesis of the phenoxazinone ring, a crucial component of the actinomycin D molecule. Glucose repression of antibiotic biosynthesis is a documented phenomenon, as reported by Sanchez and Demain (2002). Streptomyces spp. are obligate aerobes, requiring optimal aeration for growth and metabolism (Stanbury et al., 1984). This study employed a constant cultivation condition of 32°C and 200 rpm shaking for 21 days. However, fermentor studies have demonstrated that increasing agitation rate can enhance actinomycin D yield, with a maximum observed at 600 rpm (Praveen et al., 2008b). This improvement is attributed to enhanced mixing of medium components and oxygen, leading to a higher metabolic rate. Agitation exceeding 600 rpm can negatively impact production due to mycelial shearing. By optimizing both nutrient composition and bioreactor operation parameters (agitation and aeration), Praveen et al. (2008b) achieved a 356% increase in actinomycin D yield compared to shake flask cultures using a non-optimized medium. These findings suggest that further bioprocess optimization is warranted to improve actinomycin D yield from Streptomyces parvulus AL036, and achieve commercial feasibility.