Algal-mediated synthesis of nanoparticles (NPs) is an eco-friendly alternative for producing NPs with potent physicochemical and biological properties. Microalgae represent an ideal bio-nanofactory because they contain several biomolecules acting as passivation and stabilising agents during the biogenesis of NPs. Herein, a novel microalgae sp. was isolated, purified, and identified using light and electron microscopy and 18s rRNA sequencing. The chemical components of their watery extract were assessed using GC-MS. Their dried biomass was used to synthesise silver (Ag) NPs with different optimisation parameters. Ag-NPs were physiochemically characterised, and their anticancer and antibacterial effects were examined. The data showed that the isolated strain was 99% similar to the unicellular ulvophyte sp. MBIC10591; it was ellipsoidal to spherical and had a large cup-shaped spongiomorph chloroplast. The optimum parameters for synthesising Ag-NPs by unicellular ulvophyte sp. MBIC10591 (Uv@Ag-NPs) were as follows: mixture of 1 mM of AgNO₃ with an equal volume of algal extract, 100 °C for 1 h, and pH of 7 under illumination for 24 h. TEM, HRTEM, and SEM revealed that Uv@Ag-NPs are cubic to spherical, with an average nanosize of 12.1 ± 1.2 nm. EDx and mapping analysis showed that the sample had 79% of Ag, while FTIR revealed the existence of several functional groups on the NP surface derivatives from the algal extract. The Uv@Ag-NPs had a hydrodynamic diameter of 178.1 nm and a potential charge of −26.7 mV and showed marked antiproliferative activity against PC3, MDA-MB-231, T47D, and MCF-7, with IC₅₀ values of 27.4, 20.3, 23.8, and 40 µg/mL, respectively, and moderate toxicity against HFs (IC₅₀ of 13.3 µg/mL). Uv@Ag-NPs also showed marked biocidal activity against Gram-negative bacteria. Escherichia coli was the most sensitive bacteria to the NPs with an inhibition zone of 18.9 ± 0.03 mm. The current study reports, for the first time, the morphological appearance of the novel unicellular ulvophyte sp., MBIC10591, and its chemical composition and potential to synthesise Uv@Ag-NPs with smaller sizes and high stability to act as anti-tumour and microbial agents.