Aim: Marine ecosystem has served as an important source of economically and biotechnologically significant secondary metabolites. In view of this, the present study was carried out to isolate marine actinomycetes from rhizosphere soil sample and screen them for anti-microfouling activity. Methodology: Screening of marine actinomycetes was done by agar well diffusion method and microtiter plate assay. The isolates with potent anti-microfouling activity were characterized by biochemical test and scanning electron microscopy. Finally, the isolates were identified by 16S rRNA gene sequencing. The isolate VITKV6A showing maximum activity was subjected to mass cultivation and extraction of bioactive compounds using different solvents. The crude extract, thus, obtained was analyzed by GC-MS. Results: Forty marine actinomycetes were isolated from rhizosphere soil sample of Rhizophora apiculata. Three isolates VITKV6A, VITKV7A and VITKV10A showing maximum activity were selected and subjected to morphological, cultural and molecular characterization. These isolates were identified as Streptomyces thermolineatus, Streptomyces variabilis and Streptomyces althioticus by 16S rDNA sequencing. When tested for anti-microfouling activity, the chloroform extract of VITKV6A showed highest activity against all the three biofilm forming bacteria Psychrobacter celer, Psychrobacter alimentarius and Kocuria rhizophila used in this study. Gas chromatography-mass spectroscopic analysis of crude secondary metabolite extract from Streptomyces thermolineatus sp. (VITKV6A) was carried out and the results of the mass spectrum of compounds showed matches that were phenolic/polar molecules. Interpretation: The results obtained indicate that bioactive compounds from marine actinomycetes would serve as potential anti-microfouling agent for control of microfouling caused in submerged environment. © Triveni Enterprises, Lucknow (India)