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Collagen-NPs were bio-fabricated from pure marine collagen using the cell-free supernatant of a newly isolated strain, <jats:italic>Streptomyces<\/jats:italic> sp. strain NEAA-3. <jats:italic>Streptomyces<\/jats:italic> sp. strain NEAA-3 was identified as <jats:italic>Streptomyces plicatus<\/jats:italic> strain NEAA-3 based on its cultural, morphological, physiological properties and 16S rRNA sequence analysis. The sequence data has been deposited under accession number OR501412.1 in the GenBank database. The face-centered central composite design (FCCD) was used to improve collagen-NPs biosynthesis. The maximum yield of collagen-NPs was 9.33\u00a0mg\/mL with a collagen concentration of 10\u00a0mg\/mL, an initial pH of 7, an incubation time of 72\u00a0h, and a temperature of 35\u00a0\u00b0C. Using the desirability function approach, the collagen-NPs biosynthesis obtained after FCCD optimization (9.53\u00a0mg\/mL) was 3.92 times more than the collagen-NPs biosynthesis obtained before optimization process (2.43\u00a0mg\/mL). The TEM analysis of collagen-NPs revealed hollow sphere nanoscale particles with an average diameter of 33.15\u2009\u00b1\u200910.02 nm. FTIR spectra confirmed the functional groups of the collagen, collagen-NPs and the cell-free supernatant that are essential for the efficient capping of collagen-NPs. The biosynthesized collagen-NPs exhibited antioxidant activity and\u00a0anticancer activity against HeP-G2, MCF-7 and HCT116 cell lines. Collagen-NPs assessed as an effective drug loading carrier with methotrexate (MTX), a chemotherapeutic agent. The TEM analysis revealed that the average size of MTX-loaded collagen-NPs was 35.4\u2009\u00b1\u20098.9 nm. 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