The global threat of antibiotic resistance necessitates the discovery of novel antibiotics to combat drug-resistant bacterial pathogens. Actinomycetes, known for their bioactive compound production, offer a promising solution. This study evaluated the growth of Actinomycetes from Winam Gulf of Lake Victoria shores on various media, screened them for antibacterial activities, and characterized bioactive species using morphological, Biochemical and molecular markers. Eight sampling sites (Mbita, Asembo Bay, Kunya Beach, Kendu Bay, Usenge, Luanda Kotieno, Homa Bay, and Kisumu) along Winam Gulf were randomly selected. Systematic random sampling identified three sampling points (1 m in-shore) from a random starting point, with 10 m interval between points. Soil sediments were collected in triplicate from each site, giving a total of 24 samples, and transported to JOOUST Botany Laboratory. In-vitro cultural growth was assessed on Yeast Malt Extract Agar (YMEA), Soya Casein Agar, Peptone B Agar, and Yeast Extract Agar using serial dilution method. Colony size was measured on day 10. Primary screening of antibacterial activity of 16 pure isolates was done on Mueller Hinton Agar against Staphylococcus spp., Streptococcus spp., Shigella spp., Xanthomonas spp., and E. coli obtained from Kenya Medical Research Institute, Kisumu. Actinomycetes isolates were characterized morphologically, biochemically, and molecularly. Data on growth and antibacterial activity were analyzed using SAS version 21 with one-way ANOVA at 95% confidence level and means separated by Least Significant Difference (p≤0.05). Sequences were assembled using BioEdit software aligner and transferred to MEGA Version 6.0 and aligned using CLUSTAL W. Individual consensus sequences of the 16S rRNA gene regions were used to evaluate closely related sequences at the NCBI GenBank using BLAST. YMEA showed the highest growth with isolate A3 from Asembo Bay at 14.67 mm. Out of 16 pure isolates, 9 exhibited bioactivities against test pathogens. Isolate MT3 (Streptomyces microflavus) from Mbita demonstrated the highest antibacterial activity. Molecular characterization revealed limited genetic diversity among Winam Gulf Actinomycetes, predominantly within the Streptomyces genus, including Streptomyces microflavus (MT3 and KB3), Streptomyces celluloflavus (U3), Streptomyces werraensis (K3), and Streptomyces cellulosae (KSM3). Isolates MT3 and K3 are recommended for further research due to their potential in producing potent drugs against pathogenic bacteria. This study's findings contribute to academic knowledge and practical applications in combating antibiotic resistance, addressing a critical global health challenge.