Advancing microbial bioprospecting is crucial for discovering novel sources of antimicrobial compounds (ACs). Members of the phylum Actinomycetota, particularly the genus Streptomyces, are renowned producers of a wide variety of bioactive metabolites, including several clinically relevant antibiotics. In the present study, 182 Brazilian actinomycete strains were screened for antimicrobial activity against major human pathogens, namely Staphylococcus aureus MRSA BMB9393, Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 11229, Aspergillus niger ATCC 16404, Candida albicans ATCC 10231, and Cryptococcus neoformans T1444. Among these, strain AM6-12 exhibited strong and broad-spectrum inhibitory activity. To optimize its metabolite production, AM6-12 was grown in various media (ISP-1, ISP-2, Mueller–Hinton, and glycerol-peptone broth) under submerged fermentation at 28 °C for 12 days. Supernatants were collected through centrifugation and filtration and tested via disk diffusion with 100, 150, and 200 μL volumes. Inhibition zones were measured after 36 h at 35 °C. Further experiments in ISP-2 medium under varying aeration conditions (180 vs. 210 rpm; different flask volumes) showed improved activity at 180 rpm, notably against MRSA (2.6 cm) and P. aeruginosa (1.25 cm), indicating oxygen availability influences metabolite synthesis. Genomic sequencing revealed a 7.88 Mb genome with a G + C content of 72.0 mol%. The 16S rRNA gene shared 100% identity with Streptomyces malaysiense MUSC 136, but multilocus sequence analysis (MLSA) of 16S rRNA, atpD, gyrB, rpoB, recA, and trpB showed divergence above the species threshold (MLSA distance > 0.007). Additionally, ANI (< 95%) and digital DNA–DNA hybridization (dDDH < 70%) supported its classification as a distinct species. These findings position AM6-12 as a promising novel Streptomyces species for antimicrobial production.