Biofilm-associated bacterial infections pose a significant challenge in clinical settings due to their persistence and reduced susceptibility to current antimicrobials, underscoring the need for novel antibiofilm agents. This study aimed to evaluate the antibacterial and antibiofilm activities of cell-free extracts from marine Streptomyces sp. using integrated in vitro and in silico approaches. Initial antibacterial screening by agar well diffusion assay identified VITNABS17 as the most active isolate, exhibiting a broad antibacterial spectrum against the tested bacterial pathogens. Evaluation of biofilm formation by tube method revealed K. pneumoniae as the strongest biofilm-forming pathogen. The ethyl acetate extract of VITNABS17 reduced K. pneumoniae biofilm biomass by 66.2 ± 0.22% at 20 mg/mL, while the minimum biofilm inhibitory concentration (MBIC) was 1 mg/mL, corresponding to 55.50% inhibition. GC-MS analysis revealed a diverse metabolite profile, with diethyl phthalate identified as the most abundant compound. Molecular docking predicted binding affinities of −6.0 kcal/mol for MrkA and −4.4 kcal/mol for LuxS, indicating potential interactions of diethyl phthalate with biofilm-associated protein targets. The potential isolate was identified as Streptomyces sp. VITNABS17 by 16S rRNA gene sequencing. Overall, these findings provide preliminary evidence for the antibiofilm potential of VITNABS17 and warrant further investigation into the identification and characterization of its active metabolites.
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