The bacteria resident in sponges collected from Ha Tien Sea, Kien Giang province, Vietnam were investigated using the 16S rDNA gene method and investigated in vitro antimicrobial activity of marine sponge-associated bacteria. One-hundred and one bacterial isolates were isolated on two media (51 isolates on MMA medium and 50 on SYP-SW medium), 19/101 isolates had antimicrobial activity against to Edwardsiella ictaluri (fish pathogenic bacteria) and Bacillus cereus (human pathogenic bacteria). Eighteen isolates were chosen to identify with PCR-16S rDNA method, DNA sequencing were compared with GenBank database of NCBI by BLAST N software. The sequences from selected bacterial isolates (17 isolates) showed high degrees of similarity to those of the GenBank reference strains (between 99% and 100%). From 17 isolates, 16 belonged to Bacilli, and only one isolate was Beta-Proteobacteria, respectively. Based on Pi value (nucleotide diversity), Bacili group had the highest theta value and Theta values (per sequence) from S of SNP for DNA polymorphism were calculated for each group and Bacili group had the highest values. From these results showed that two strains (Bacillus subtilis MD4B and Bacillus thuringiensis SL1B) revealed promising candidates because they have the potential bioactivity, either against fish pathogenic bacteria as Edwardsiella ictaluri or fish and human pathogens as E. ictaluri and Bacillus cereus, they will be suggested to evaluate at high scale in the future.