The bacteria associated with living surfaces are rich sources of bioactive metabolites. In the present study, 182 heterotrophic epibacterial colonies, isolated from seaweeds (44%), ascidians (30.2%), barnacles (10.4%) and molluscan egg mass (15.4%), were subjected to high throughput screening by cross streaking method against six human pathogenic bacteria, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Vibrio cholerae, Proteus mirabilis and Klebsiella pneumoniae. The 137 epibacterial isolates, which showed activity against at least one human bacterial pathogen in cross streaking method, were further cultured and the ethyl acetate extract of the culture broth (100 μg/disc) was assayed for antibacterial activity through disc diffusion method. The four epibacterial colonies, BR1, EM13, EM14 and PC4, isolated from the barnacle Balanus amphitrite, seaweed Enteromorpha compressa, and ascidian Polyclinum constellatum showed broad spectral antibacterial activity.