Thiolutin and 2,2-dioxidothiolutin are sulfur-containing antibiotics discovered through a targeted high-throughput screening strategy designed to identify compounds with antimicrobial activity. The producing strain, JHD1, is an actinomycete isolated from marine sediment collected in Chonburi Province, Thailand. Chemotaxonomic characteristics and 16S rRNA gene sequence analysis indicated that strain JHD1 belongs to the genus Streptomyces. Taxonomic analysis using a polyphasic approach further supported its placement within the genus Streptomyces. The strain exhibited low similarity to its closest known relatives, Streptomyces alkaliterrae OF1 and Streptomyces chumphonensis KK1-2, with average nucleotide identity based on MuMmer ultra-rapid aligning tool (ANIm) values of 85.7-86.2%, average amino acid identity (AAI) values of 71.8-78.1%, and digital DNA-DNA hybridization (dDDH) values of 23.4-25.0%, all well below the established thresholds for species delineation. These results reveal that strain JHD1 represents a novel species within the genus Streptomyces, for which the name Streptomyces marinisediminis sp. nov. is proposed. The crude extract obtained from the culture broth of strain JHD1 exhibited strong antimicrobial activity against Gram-positive bacteria and led to the discovery of two promising secondary metabolites, thiolutin and 2,2-dioxidothiolutin, along with 8-amino-2H-benz[e][1,3]oxazine-2,4(3H)-dione, benadrostin, 2,3-dihydroxybenzamide, and 2-hydroxy-3-methoxybenzamide. In this study, 2,2-dioxidothiolutin was structurally confirmed for the first time by X-ray crystallographic analysis. Interestingly, thiolutin demonstrated strong anti-human small cell lung cancer (NCI-H187) activity, with an IC₅₀ value of 0.35 µg/mL, and also showed cytotoxicity against human breast cancer (MCF-7) cells, with an IC₅₀ value of 5.61 µg/mL.